Freeze-dried fibrin matrices and methods for preparation thereof

ABSTRACT

Methods for treating diseased or injured tissue by implanting into the tissue at a site of the disease or injury a porous freeze-dried fibrin matrix formed from plasma proteins. The proteins include fibrinogen cleaved by the action of thrombin at varying concentrations sufficient to cleave the fibrinogen and Factor XIII. The matrix has less than 10% residual moisture and is devoid of exogenous anti-fibrinolytic agents, plasminogen and of organic chelating agents. Alternatively, the plasma proteins comprise partially purified plasma proteins that are devoid of plasminogen.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a division of application Ser. No. 12/731,356 filedMar. 25, 2010, which is a continuation of application Ser. No.11/190,387 filed Jul. 26, 2005, now U.S. Pat. No. 7,714,107, which is acontinuation of International application PCT/IL2004/000088 filed Jan.29, 2004, which claims the benefit of provisional application 60/507,167filed Oct. 1, 2003, the entire content of each of which is expresslyincorporated herein by reference thereto.

FIELD OF THE INVENTION

The present invention concerns freeze-dried biomatrices comprisingplasma proteins substantially devoid of plasminogen useful for clinicalapplications including as implants for tissue engineering. Additionalpreferred embodiments of the invention are freeze-dried plasma proteinbiomatrices substantially devoid of exogenous anti-fibrinolytic agents.The matrices according to the present invention are useful clinically,per se or as cell-bearing implants.

BACKGROUND OF THE INVENTION Tissue Engineering

Tissue engineering may be defined as the art of reconstructing orregenerating mammalian tissues, both structurally and functionally(Hunziker, Osteoarth. Cart. 10: 432-63, 2002). Tissue engineeringgenerally includes the delivery of a synthetic or natural scaffold thatserves as an architectural support onto which cells may attach,proliferate, and synthesize new tissue to repair a wound or defect.

An example of a tissue that is prone to damage by disease and trauma isthe articular cartilage, one of several types of cartilage in the body,found at the articular surfaces of bones. Damage to cartilage may resultfrom an inflammatory disease such as rheumatoid arthritis, from adegenerative process such as osteoarthritis or from trauma such asintraarticular fracture or following ligament injuries. Cartilagelesions are often associated with pain and reduced function, generallydo not heal and without medical intervention may require total jointreplacement.

Current therapeutic strategies for repairing damaged cartilage encompassprocedures that induce a spontaneous repair response and those whichreconstruct the tissue in a structural and functional manner. The formerincludes surgical techniques that expose the subchondral bone therebyallowing the infiltration of bone marrow progenitor cells to initiatethe healing response. Often the induced tissue is of a mixedfibrocartilage type, is not durable and the clinical improvements areshort lived. The latter strategy includes transplantation of chondral orosteochondral cells or tissue from either an autologous or an allogeneicsource. Autologous Chondrocyte Transplantation (ACT) relies ontransplanting into a cartilage lesion autologous chondrocytes, whichhave been isolated from a patient's cartilage biopsy and expanded invitro.

In fact, this technique requires a complicated procedure involving twosurgical sites, and shows high variability and limited clinical success.

Matrices useful for tissue regeneration and/or as biocompatible surfacesuseful for tissue culture are well known in the art. These matrices maytherefore be considered as substrates for cell growth either in vitro orin vivo. Suitable matrices for tissue growth and/or regeneration includeboth biodegradable and biostable entities. Among the many candidatesthat may serve as useful matrices claimed to support tissue growth orregeneration are gels, foams, sheets, and porous structures of differentforms and shapes.

Porous materials formed from synthetic and/or naturally occurringbiodegradable materials have been used in the past as wound dressings orimplants. A porous material provides structural support and a frameworkfor cellular in-growth and tissue regeneration. Preferably, the porousmaterial gradually degrades and is absorbed as the tissue regenerates.Typical bioabsorbable materials for use in the fabrication of porouswound dressings or implants include both synthetic polymers andbiopolymers such as structural proteins and polysaccharides. Thebiopolymers may be either selected or manipulated in ways that affecttheir physico-chemical properties to provide greater or lesser degreesof flexibility or susceptibility to degradation.

Many natural polymers have been disclosed to be useful for tissueengineering or culture, including various constituents of theextracellular matrix including fibronectin, various types of collagen,and laminin, as well as keratin, fibrin and fibrinogen, hyaluronic acid,heparan sulfate, chondroitin sulfate and others. U.S. Pat. Nos.6,425,918 and 6,334,968 disclose a freeze-dried bioresorbablepolysaccharide sponge and use thereof as a matrix or scaffold forimplantation into a patient.

Fibrin

Fibrinogen is a major plasma protein, which participates in the bloodcoagulation process. Upon blood vessel injury, fibrinogen is convertedto insoluble fibrin which serves as the scaffold for a clot. Bloodcoagulation of is a complex process comprising the sequentialinteraction of a number of plasma proteins, in particular of fibrinogen(factor 1), prothrombin (factor II), factor V and factors VII-XIII.Other plasma proteins such as Von Willebrand factor, immunoglobulins,coagulation factors and complement components also play a part in theformation of blood clots.

Fibrin is known in the art as a tissue adhesive medical device usefulfor wound healing and tissue repair. Lyophilized plasma-derived proteinconcentrate (comprising fibrinogen, Factor XIII and fibronectin), in thepresence of thrombin and calcium ions forms an injectable biologicalsealant (fibrin glue). U.S. Pat. No. 5,411,885 discloses a method ofembedding and culturing tissue employing fibrin glue.

U.S. Pat. No. 4,642,120 discloses the use of fibrinogen-containing gluein combination with autologous mesenchymal or chondrocytic cells topromote repair of cartilage and bone defects. U.S. Pat. No. 5,260,420discloses a method for preparation and use of biological glue comprisingplasma proteins for therapeutic use. U.S. Pat. No. 6,440,427 provides anadhesive composition consisting substantially of fibrin formingcomponents and a viscosity enhancing polysaccharide such as hyaluronicacid. A freeze-dried fibrin clot for the slow release of an antibioticis described by Itokazu (Itokazu et al., Infection 25: 359-63, 1997).U.S. Pat. No. 5,972,385 discloses a lyophilized crosslinkedcollagen-polysaccharide matrix, with optional fibrin, that isadministered per se or in combination with therapeutics for tissuerepair. U.S. Pat. Nos. 5,206,023 and 5,368,858 disclose a method andcomposition for inducing cartilage repair comprising dressing the sitewith a biodegradable matrix formed by mixing matrix forming materialwith a proliferative agent and a transforming factor.

A fibrinogen-containing freeze-dried fleece-like structure for use as awound dressing, filling for bone cavities or support material forrelease of active materials has been disclosed in U.S. Pat. No.4,442,655. The structure is prepared by premixing fibrinogen andthrombin solutions, pouring into a mold, freezing and lyophilizing.

A freeze-dried fibrin web for wound healing has been disclosed in U.S.Pat. Nos. 6,310,267 and 6,486,377. The preparation of said webnecessitates a single- or multi-stage dialysis of the fibrinogensolution. According to that disclosure, the single-stage or multistagedialysis of the fibrinogen solution changes crucially its composition byreducing the concentration of salts and amino acids. The dialysis iscarried out in an aqueous solution of a physiologically compatibleinorganic salt and an organic complexing agent.

A storage stable fibrin sponge containing a blood clotting activator forhemostasis, tissue adhesion, wound healing and cell culture support isdisclosed in WO 99/15209. According to that disclosure, the restorationof moisture or water content following lyophilization is crucial forobtaining a soft, adaptable, absorbent sponge. The sponge may beimpregnated with additives such as a blood clotting activator,stabilizers, preservatives and other agents.

U.S. Pat. Nos. 5,466,462 and 5,700,476 disclose a bioresorbableheteromorphic sponge comprising a biopolymer matrix structure, at leastone substructure and at least one pharmacologically active agent. Thesubstructures allow the incorporation of one or more active agents intothe final product for physic release. U.S. Pat. No. 5,443,950 relates tothe growth of cells derived from a desired tissue on a pre-establishedstromal support matrix. U.S. Pat. No. 5,842,477 discloses a method of invivo cartilage repair by implanting a biocompatible, three-dimensionalscaffold in combination with periosteal/perichondrial tissue and stromalcells, with or without bioactive agents.

Fibrinolysis

Existing freeze-dried fibrin implants for tissue engineering purposesare prepared using fibrinogen or plasma protein solutions havinginherent proteases that may compromise the stability of certain of theplasma proteins and lead to degradation of the matrix. Plasminogen is amajor plasma protein that binds fibrin during clot formation.

Within the clot or matrix, plasminogen is enzymatically converted toplasmin, which functions as a fibrinolytic agent, resulting in thedegradation of the clot or matrix. This process is typically retarded bythe addition of anti-fibrinolytic agents, including but not limited toaprotinin, s-aminocaproic acid or tranexamic acid into the composition.These agents may have detrimental effects on cell growth, proliferationand/or differentiation or may cause adverse reactions in patients. Theart has not heretofore provided a stable freeze-dried fibrin matrixsubstantially devoid of exogenous anti-fibrinolytic agents.

Copending international patent application WO 03/007873 by some of theapplicants of the present invention of the present invention, disclosesa freeze-dried plasma protein matrix comprising plasma proteins and atleast one anti-fibrinolytic agent, optionally comprising selectedauxiliary agents to improve certain physical, mechanical and biologicalproperties of the matrix.

Thus, there remains an unmet need for a fully biocompatible, truethree-dimensional, plasma protein matrix, for in vitro and in vivo cellgrowth and tissue regeneration, substantially devoid of fibrinolyticactivity and exogenous anti-fibrinolytic agents thus obviating the needfor exogenous anti-fibrinolytic agents.

SUMMARY OF THE INVENTION

The present invention relates to biomatrices substantially devoid ofexternal anti-fibrinolytic agents, which have been shown to bedeleterious to cells and tissue and which may induce adverse reactionsin patients. It is now disclosed for the first time that resilient,non-brittle, matrices, also known as sponges, that are particularlybeneficial for supporting three dimensional cell growth may be obtainedfrom plasma proteins substantially devoid of plasminogen, thus obviatingthe need for external anti-fibrinolytic agents. It is further disclosedthat unexpectedly biomatrices obtained from partially purified plasmaproteins also obviates the need for exogenous anti-fibrinolytic agents.The compositions and methods of the present invention are effective forin vivo and in vitro applications including as biocompatible implantsfor tissue engineering as well as in biotechnology for the in vitroculturing and differentiation of cells. The matrices according to thepresent invention are three-dimensional (3D) and may be used clinically,per se or as cell-bearing implants. The present invention provides allcomponents fundamental for tissue repair, thus facilitating the medicalpractitioner's task and providing a superior alternative for tissuereparation and regeneration in a patient.

The present invention provides a porous, freeze-dried fibrin matrixformed from plasma proteins comprising fibrinogen, thrombin and FactorXIII, the matrix having less than 10% residual moisture and beingsubstantially devoid of exogenous anti-fibrinolytic agents and oforganic chelating agents, which exhibits superior characteristics. Thepresent invention is based in part on the unexpected finding that amatrix comprising plasma proteins substantially devoid of exogenousanti-fibrinolytic agents and plasminogen imparts superior stability andsignificantly improves cell seeding and cell dispersion while retainingother positive attributes of these matrices. The plasminogen-freematrices in particular exhibit reduced resorbability compared to fibrinmatrices known in the art, providing a long lasting implant withenhanced stability and endurance for successful tissue growth, repairand regeneration.

In one aspect, the present invention relates to a porous freeze-driedfibrin matrix formed from plasma proteins substantially devoid ofexogenous anti-fibrinolytic agents and of organic chelating agents.According to one embodiment the plasma proteins are purified from aplasma source or may be used from a commercially available source,including native or recombinant proteins, in the substantial absence ofexogenous anti-fibrinolytic agents and of organic chelating agents.According to another embodiment the plasma protein source is selectedfrom total blood, blood fractions, blood derivative, cryoprecipitate,recombinant proteins, plasma and plasma fractions. The plasma proteinsmay be selected from xenogeneic, allogeneic and autologous plasmasources. According to one embodiment the plasma source is autologous.

In another aspect, the present invention provides a porous freeze-driedfibrin matrix formed from plasma proteins comprising fibrinogen,thrombin and Factor XIII, the matrix having less than 10% residualmoisture and being substantially devoid of exogenous anti-fibrinolyticagents, plasminogen and of organic chelating agents. In one embodimentsubstantially devoid of plasminogen refers to the plasma proteinsolution comprising less than about 20% of plasminogen normally presentin blood plasma, preferably less than about 10% of the plasminogennormally present in plasma and more preferably less than about 5% of theplasminogen normally present in plasma. The inventors have discoveredthat a porous freeze-dried fibrin matrix comprising plasma proteinssubstantially devoid of plasminogen provides a superior matrix forclinical and biotechnological applications. In addition to eliminatingthe need for exogenous anti-fibrinolytic agents and their concomitantdetrimental effects, the inventors now show that the fibrin matrix ofthe present invention is superior as a scaffold for cell seeding, growthand differentiation and for use in tissue repair and regeneration.

The fibrin matrix of the invention may be used per se, comprising plasmaproteins substantially devoid of exogenous anti-fibrinolytic agents andof organic chelating agents, for clinical and biotechnologicalapplications. It may however, further comprise additives that impartadditional advantageous biological, physical and mechanicalcharacteristics to the matrix. The present invention encompasses theincorporation into the matrix of at least one additive to provide amatrix having improved biological, mechanical and/or physicalproperties.

Copending international patent application WO 03/007873 by some of theapplicants of the present invention discloses a fibrin matrix comprisingplasma proteins and at least one anti-fibrinolytic agent, optionallyfurther comprising agents such as polysaccharides, anionicpolysaccharides, glycosaminoglycans, or synthetic polymers added in thepreparation to improve certain physical, mechanical and biologicalproperties of the matrix. The requirement for an anti-fibrinolytic agenthas now been removed or overcome by the substantial absence ofplasminogen in the matrix.

In one embodiment, the present invention is related to a porous fibrinmatrix substantially devoid of exogenous anti-fibrinolytic agents,plasminogen and of organic chelating agents further comprising at leastone additive selected from the group consisting of polysaccharides,glycosaminoglycans (GAGs) and synthetic polymers that is useful as asupport for growth and differentiation of cells, both in vitro and invivo.

According to one embodiment the additive may be added ab initio, i.e.,during formation of the clot. According to alternative embodiments theadditive is introduced to the matrix any time following formation of thematrix. According to various embodiments of the present invention, thematrix is prepared using at least one glycosaminoglycan selected fromthe group consisting of crosslinked hyaluronic acid, non-crosslinkedhyaluronic acid, heparin and heparin derivatives and heparin mimetics,chondroitin sulfate, dextran sulfate, dermatan sulfate, heparan sulfateand keratan sulfate.

The glycosaminoglycan is added to the matrix at a final concentrationthat imparts suppleness and elasticity to the matrix and precludes theneed for adjusting the moisture content of the final composition.According to one embodiment of the present invention theglycosaminoglycan is selected from crosslinked and non-crosslinkedhyaluronic acid. In one embodiment the concentration of non-crosslinkedhyaluronic acid is about 0.005% to about 0.5% final (V/V) morepreferably about 0.05% to about 0.1%. In another embodiment theconcentration of crosslinked hyaluronic acid is about 0.001% to about0.1% and more preferably about 0.05% to about 0.09% final (V/V).According to another embodiment the glycosaminoglycan is selected fromheparin and heparin derivatives.

The present invention further encompasses a fibrin matrix comprising atleast one bioactive agent selected from the group consisting oftherapeutic proteins, platelets and platelet supernatant, analgesics,anti-microbial or anti-inflammatory agents and enzymes.

According to one embodiment the present invention provides afreeze-dried porous matrix comprising plasma proteins substantiallydevoid of exogenous anti-fibrinolytic agents, plasminogen and of organicchelating agents, further comprising at least one glycosaminoglycan andat least one bioactive agent.

According to another embodiment of the present invention the at leastone bioactive agent is a therapeutic protein selected from the groupconsisting of growth factors and their variants. In one aspect, thegrowth factor is selected from a fibroblast growth factor (FGF) andvariants thereof. In another aspect, the FGF is an FGF having thecapacity to induce or enhance cartilage and bone repair and regenerationand or angiogenesis. The growth factors may be incorporated at a widerange of concentrations, depending on the potency of the factor and theintended application.

For certain applications, sustained or phasic release of a bioactiveagent may be preferred. In one embodiment, the at least one growthfactor is incorporated in to the fibrin matrix directly, ab initio. Inanother embodiment, the at least one growth factor is bound to a carriermolecule such as heparin and is incorporated into the matrix ab initio.Sustained release of a bioactive agent depends on several factorsincluding growth factor concentration, type of glycosaminoglycanincorporated and fibrin and thrombin concentration.

In contrast to the bioabsorbable heteromorphic sponge of the art, thepresent inventors now disclose a freeze-dried fibrin sponge compromisingplasma proteins substantially devoid of exogenous anti-fibrinolyticagents, plasminogen and of organic chelating agents optionallycomprising at least one additive selected from the group consisting ofpolysaccharides, glycosaminoglycans and synthetic polymers andoptionally further comprising at least one bioactive agent.

According to one embodiment, the present invention provides a porousfreeze-dried fibrin matrix comprising plasma proteins substantiallydevoid of exogenous anti-fibrinolytic agents, plasminogen and of organicchelating agents, further comprising at least one glycosaminoglycan andat least one bioactive agent. According to another embodiment of theinvention the at least one glycosaminoglycan is selected from heparinand derivatives thereof, the at least one bioactive agent is atherapeutic protein selected from the FGF family of growth factors andvariants thereof. This sponge provides phasic release of the FGF fromthe matrix and may be beneficial in certain therapeutic applications.

According to another embodiment the present invention provides a porousfreeze-dried fibrin matrix comprising plasma proteins substantiallydevoid of exogenous anti-fibrinolytic agents, plasminogen and of organicchelating agents further comprising hyaluronic acid, heparin and atleast one bioactive agent. The hyaluronic acid is selected fromcrosslinked and non-crosslinked hyaluronic acid. Preferably, thehyaluronic acid and the heparin or heparin derivative are incorporatedinto the sponge ab initio. The bioactive agent such as a growth factormay be incorporated into the sponge per se or heparin bound. Preferablythe growth factor is selected from the family of FGF therapeuticmolecules.

Another aspect of the invention provides a method of preparing theporous fibrin matrix. A method for preparing a porous freeze-driedfibrin matrix having less than 10% residual moisture and beingsubstantially devoid of exogenous anti-fibrinolytic agents and oforganic chelating agents comprises the following steps:

-   -   providing a thrombin solution and a plasma protein solution        wherein the plasma protein solution is substantially devoid of        exogenous anti-fibrinolytic agents and of organic chelating        agents;    -   introducing the thrombin solution and the plasma protein        solution to a solid receptacle or mold in the presence of        calcium ions; incubating under conditions appropriate to achieve        clotting;    -   freezing the clotted mixture; and    -   lyophilizing the clotted mixture, to obtain a sponge, and    -   optionally seeding the sponge with cells prior to implantation.

According to one embodiment of the present invention the plasma proteinsare partially purified plasma proteins. According to another embodimentof the present invention the plasma proteins are devoid of plasminogen.According to yet another embodiment the plasma protein solutioncomprising less than about 20% of plasminogen normally present in bloodplasma, preferably less than about 10% of the plasminogen normallypresent in plasma and more preferably less than about 5% of theplasminogen normally present in plasma.

According to one embodiment of the invention the porous fibrin sponge isprepared by transferring the thrombin solution into a mold or solidreceptacle, adding the plasma protein solution to achieve clotformation; freezing the clotted mixture and lyophilizing. Alternatively,the plasma proteins are mixed with thrombin in the presence of calciumions under conditions suitable for achieving clotting; the mixture iscast in a solid support prior to achieving clotting; the clotted mixtureis frozen and lyophilized. It is to be understood that whenincorporated, additives and bioactive agents are added independently ofeach other to either of the matrix forming solutions, i.e. the plasmaproteins or the thrombin solution, prior to the formation of the clot orare placed into the mold or solid receptacle prior to, concurrently withor following addition of the thrombin.

In one embodiment the invention provides a heterogeneous porous fibrinmatrix wherein particulate matter is incorporated into the sponge abinitio. Particulate matter may include materials such as calciumphosphate particles, bone chips or glass fibers that are able to impartcertain advantageous properties to the matrix including strength,additional porosity or phasic release.

According to various embodiments of the present invention plasmaproteins at a concentration of about 10 mg/ml to about 50 mg/ml,substantially devoid of anti-fibrinolytic agents, plasminogen and oforganic chelating agents, are mixed with at least one glycosaminoglycansuch as hyaluronic acid and/or heparin, the mixture is incubated andadded to the thrombin solution in the solid support to achieve formationof a clot. The clot is subsequently frozen and lyophilized.

In one embodiment, prior to implantation or use with cells, the spongeis substantially dry and contains less than 15% residual moisture,preferably less than 10% residual moisture. Surprisingly, this propertyof the sponge has been shown to be particularly advantageous for cellseeding and attachment.

Another aspect of the present invention provides a method of treatmentand use of the freeze-dried fibrin matrix substantially devoid ofexogenous anti-fibrinolytic agents and of organic chelating agents fortissue regeneration and repair of injured, diseased or traumatizedtissue, including cartilage and bone defects and other tissue typesincluding but not limited to liver, pancreas, and cardiac tissue. Themethod of treatment described herein is advantageous in that it requiresminimal preparation for use by the medical practitioner. Otheradvantageous properties derive from the absence of exogenousanti-fibrinolytic agent such as tranexamic acid and aprotinin, which maybe detrimental to the patient and the tissue surrounding the implant.Additionally, the absence of an exogenous anti-fibrinolytic agentrenders the sponge a superior scaffold for in vivo or in vitro cellularattachment, growth, proliferation, infiltration and differentiation.

According to one embodiment the sponge is implanted per se. In anotherembodiment the sponge is cut into at least one section of desired shape.

In one embodiment the sponge further comprises cells. According toanother embodiment the cells are selected from stem cells or progenitorcells. According to yet another embodiment the cells are selected fromchondrocytes, osteoblasts, hepatocytes, or mesenchymal, endothelial,epithelial, urothelial, endocrine, neuronal, pancreatic, renal andocular cell types.

The in vivo uses of the porous fibrin matrix are manifold. The porousfibrin matrix may function as a scaffold for in vitro culturing of cellsor as an implant per se, for providing mechanical support to a defectiveor injured site in situ and/or for providing a matrix within which cellsfrom the defective or injured site invade, proliferate and/ordifferentiate. The matrix is useful in treating articular cartilagedefects of any type, including chondral and subchondral defects, arisingfrom trauma such as an accident or sports injury or disease such asosteoarthritis. The porous fibrin matrix may be used per se or incombination with other therapies. For example, for cartilage repair theporous fibrin matrix is useful in conjunction with other therapeuticprocedures including chondral shaving, laser or abrasion chondroplasty,and drilling or microfracture techniques.

Other typical orthopedic applications include joint resurfacing,meniscus repair, non-union fracture repair, craniofacial reconstructionor repair of an invertebral disc. Furthermore, the porous fibrin matrixis useful as a coating on synthetic or other implants such as pins andplates, for example, in hip replacement procedures. Thus, the presentinvention further provides implants or medical devices coated with afinish comprising the porous fibrin matrix of the invention.

The porous fibrin matrix of the invention is useful, inter alfa, as anunexpectedly advantageous support for cellular growth. The absence ofexogenous anti-fibrinolytic agents results in a fibrin matrix that isfully compatible with in vitro and in vivo cell growth, proliferationand differentiation. An additional advantage of the fibrin matrix of theinvention is its improved ability to absorb cells and maintain theirviability. The need to hydrate or rinse the sponge of the inventionprior to cell seeding is precluded by the absence of exogenousanti-fibrinolytic agents, thus rendering a sponge with superior cellincorporation capacity.

The porous fibrin matrix of the invention, being an effective scaffoldsupporting cell growth, may be utilized in vivo in reconstructivesurgery, for example as a matrix for regenerating tissue comprisingneuronal cells, hepatic cells, urothelial cells, osteoblasts,cardiovascular tissue and mammary tissue or any other cell types whichit is desired to culture within a three dimensional support. Thus, thematrix of this invention may be used to construct living tissueequivalents, including but not limited to liver, pancreas, nerve,glands, tendons, skin, blood vessels, bone, tendon, ligaments, and otherorgan equivalents, among many others.

According to one embodiment of the present invention the matrix is asponge or scaffoldable to support the proliferation of a variety of celltypes. In one aspect, the sponge is inoculated with cells and the cellsare allowed to proliferate in vitro prior to in vivo implantation.Alternatively, the sponge is seeded with cells that have been culturedor harvested and the sponge comprising the cells is implanted in situ.In one embodiment the porous fibrin matrix useful as an implant fortransplantation comprises autologous plasma proteins and autologouschondrocytes.

According to one embodiment the present invention provides a method oftreating or repairing injured, diseased or traumatized tissue, themethod comprising the step of implanting a porous freeze-dried fibrinmatrix formed from plasma proteins comprising fibrinogen, thrombin andFactor XIII, the matrix having less than 10% residual moisture and beingsubstantially devoid of exogenous anti-fibrinolytic agents and oforganic chelating agents to the site of injury, disease or trauma. Thetissue is selected from cartilage, bone, liver, mesenchymal,endothelial, epithelial, urothelial, endocrine, neuronal, pancreatic,renal and ocular tissue types. According to another embodiment theporous freeze-dried fibrin matrix formed from plasma proteins comprisingfibrinogen, thrombin and Factor XIII, the matrix having less than 10%residual moisture and being substantially devoid of exogenousanti-fibrinolytic agents, plasminogen and of organic chelating agents isimplanted into the site of injury disease or trauma.

Further provided is the use of an implant of the present invention forthe treatment or repair of injured, diseased or traumatized tissue, theuse comprising the step of implanting a matrix of the present inventionto the site of injury, disease or trauma. The tissue is selected fromcartilage, bone, liver, mesenchymal, endothelial, epithelial,urothelial, endocrine, neuronal, pancreatic, renal and ocular tissuetypes.

These and further embodiments will be apparent from the figures,detailed description and examples that follow.

BRIEF DESCRIPTION OF THE FIGURES

The present invention will be understood and appreciated more fully fromthe following detailed description taken in conjunction with the figuresin which:

FIG. 1A shows a graph of porcine chondrocyte viability on the matrixsubstantially devoid of plasminogen compared to a standard spongecomprising an exogenous anti-fibrinolytic agent. FIG. 1B shows humanchondrocyte viability on a matrix substantially devoid of plasminogencompared to a standard sponge comprising an exogenous anti-fibrinolyticagent. FIG. 1C shows the viability of human chondrocytes seeded onmatrices substantially devoid of plasminogen, with or without hyaluronicacid.

FIGS. 1D and 1E show pictures of the matrices of the invention, dry andseeded with cells, respectively.

FIGS. 2A-2C show histological sections of chondrocyte distribution inthe fibrin sponges substantially devoid of plasminogen, followingone-week culture.

FIGS. 3A and 3B show the rate of degradation of the sponge substantiallydevoid of plasminogen compared to a sponge comprising tranexamic acid,in urea or collagenase.

FIGS. 4A-4B represent FGF release from fibrin matrices substantiallydevoid of plasminogen comprising 0.08% crosslinked hyaluronic acid andvarying amounts of heparin prepared in two different ways. FIG. 4C showsa bar graph of FGF release from plasma protein matrices comprisingtranexamic acid (commercial) or human plasma protein matrix comprisingpartially purified plasma proteins and 0.024% hyaluronic acid and FGFand heparin incorporated, ab initio. FIG. 4D shows the FGF releasepattern from plasma protein matrices substantially devoid of exogenousanti-fibrinolytic agents comprising varying concentrations of thrombinand heparin.

FIGS. 5A-5D show histochemical sections of a plasma protein comprisingpartially purified human plasma proteins seeded with porcinechondrocytes.

FIG. 6A shows primary rat hepatocyte cells incubated for three days on aporous freeze-dried plasma protein matrix, substantially devoid ofplasminogen. FIGS. 6B and 6C show the CHO and L8 cell lines incubatedfor three days on a porous freeze-dried plasma protein matrix,substantially devoid of plasminogen.

FIG. 7A shows the insertion of a cell-bearing porous freeze-dried plasmaprotein matrix, substantially devoid of plasminogen into a subcutaneouspocket of a nude mouse. FIG. 7B shows the neocartilage that developed.

FIGS. 8A-8C show histological cross sections of a neocartilage nodule.FIG. 8A shows the cell matrix formed after 1 week, as stained withtoluidine blue and fast red.

FIGS. 8B and 8C show histological sections of the neocartilage nodulestained with H&E, magnified ×200 and ×400, respectively.

FIGS. 9A-9B show histological cross sections of the neocartilage nodulestained with H&E, at 10× and ×100 magnification.

FIG. 10A depicts cell viability on plasma protein sponges preparedeither by premixing the plasma protein and thrombin solutions or bymixing the solutions during casting. FIGS. 10B and 10C show histologicalcross sections of cell-bearing sponges substantially devoid ofplasminogen prepared by premixing the plasma protein and thrombinsolutions (10B) or by mixing the plasma protein and thrombin solutionsduring the casting step (10C).

DETAILED DESCRIPTION OF THE INVENTION

Though numerous biomatrices comprising plasma or tissue proteins areknown in the art to which the present invention pertains, none hasproven entirely satisfactory in meeting the criteria required forsuccessful tissue engineering and tissue reparation. The presentinvention discloses a porous fibrin matrix, also referred to as asponge, comprising plasma proteins substantially devoid of plasminogenand of organic chelating agents. The absence of plasminogen in thematrix obviates the need for external anti-fibrinolytic agents. It isfurther disclosed that unexpectedly biomatrices comprising partiallypurified plasma proteins also obviate the need for the addition ofexogenous anti-fibrinolytic agents. The compositions and methods of thepresent invention are effective for in vitro and in vivo applicationsincluding as cell-bearing implants for tissue engineering andreparation.

The resulting fibrin, or plasma protein, sponge has attributes that makeit particularly advantageous for supporting and promoting cell growthboth in vivo and in vitro. Plasminogen is a plasma protein which isenzymatically converted to an active serine protease, plasmin, havingfibrinolytic activity. This activity results in the rapid degradation offibrin in fibrin glue and matrices. Anti-fibrinolytic agents such astranexamic acid and aprotinin are typically incorporated into fibringlue, sponges and matrices in order to maintain the integrity of thesubstrate. The sponges of the present invention are stable and exhibitreduced bioresorbability and overcome the need to add exogenousanti-fibrinolytic agents.

Among the advantageous properties of the matrices of the invention:

The fibrin matrices exhibit superior biological properties includingreduced biodegradability, an absence of immunogenicity or other adversereactions, the capacity to maintain and promote high levels of cellgrowth, proliferation, differentiation and migration and controlledrelease of bioactive agents.

The matrices have superior mechanical properties, controlled by varyingthe additives used in the composition. Desirable properties includesuppleness, elasticity and durability.

The matrices have superior physical properties, which may be controlledby the additives used in the composition. The desirable propertiesinclude texture, pore size and interconnecting channels, hydrophilicity,hydrophilicity, adhesion, wettability, adherence and texture.

The plasma proteins can be retrieved from autologous or recombinantmaterial thereby obviating the need for pooled blood sources with theattendant health risks.

The matrices of the invention provide all components fundamental fortissue repair, thus facilitating the medical practitioner's task. Inaddition, the composition of the sponge renders it suitable forminimally invasive surgery of articular cartilage. The sponge may beimplanted in a mini-arthrotomy or arthroscopy procedure, thus avoidingthe multiple site surgeries and a full arthrotomy used for ACT.

DEFINITIONS

For convenience and clarity certain terms employed in the specification,examples and claims are described herein.

“Plasma” as used herein refers to the fluid, non-cellular portion of theblood of humans or animals as found prior to coagulation.

“Plasma protein” as used herein refers to the soluble proteins found inthe plasma of normal humans or animals. These include but are notlimited to coagulation proteins, albumin, lipoproteins and complementproteins. The major plasma protein is fibrinogen, which upon cleavage bythrombin in the presence of calcium ions and Factor XIII, is convertedto fibrin. A fibrin matrix may be used interchangeably with a plasmaprotein matrix.

As used herein the term “plasminogen” refers to plasminogen and plasmin.The terms “Substantially devoid of plasminogen” or “plasminogen-free”refer to plasma proteins having less than about 20% plasminogen normallypresent in plasma, preferably less than about 10% plasminogen normallypresent in plasma, preferably less than about 5% of the plasminogennormally present in plasma. Plasma normally compromises about 200 mgplasminogen per liter fresh plasma (about 2 μmol/liter). Plasminogen isthe precursor to the active enzyme plasmin.

A “substantial absence of organic chelating agents” or “substantiallydevoid of organic chelating agents” refers to a concentration of lessthan 1 mm of an organic chelating agent such as EDTA or other organicchelating agents known in the art.

“Substantially devoid of exogenous anti-fibrinolytic agents” or“substantially devoid of external anti-fibrinolytic agents” refer to aplasma protein or fibrinogen solution to which no anti-fibrinolyticagents have been added. Non-limiting examples of antifibrinolytic agentsinclude tranexamic acid (TEA), aprotinin and ε-aminocaproic acid (EACA).It is to be noted that small amounts of exogenous anti-fibrinolyticagents may be present in the plasma proteins due to processing methods.

“Platelet rich plasma” or “PRP” as used herein refers to plasmacontaining platelets. A platelet sample or platelet-derived extract orsupernatant may be added exogenously. Alternatively, platelet richplasma may be prepared by methods known in the art, including thosedisclosed in U.S. Pat. No. 6,475,175 and U.S. Pat. No. 6,398,972.

A “matrix” as used herein, refers to a porous structure, solid orsemi-solid biodegradable substance having pores and interconnectingchannels sufficiently large to allow cells to populate, or invade thematrix. The fibrin matrix of the invention may have irregular pores orsubstantially regular pores. As used herein, the term “substantiallyregular pores” means that the majority of the pores or more preferablysubstantially all the pores are in the same size range. Thematrix-forming materials require addition of a polymerizing agent toform the matrix, such as addition of thrombin in the presence ofbivalent calcium ions to a solution comprising fibrinogen to form afibrin clot. The clot is subsequently freeze-dried yielding a porousfibrin matrix. The fibrin matrix of the present invention may be denotedherein as a scaffold, biomatrix or as a sponge, for use as an implantper se, for the culturing of cells or as a cell-bearing tissuereplacement implant. Although the examples presented herein refer to theuse of the matrix in cartilage repair, it is to be understood that thematrix may be used for tissue reparation and regeneration of many othertissue types including bone, mammary, epithelial, neural, hepatic andendothelial tissue types.

The term “stem cell” as referred to herein refers to an undifferentiatedcell that is capable of proliferation. Stem cells are capable ofproducing either new stem cells or cells called “progenitor cells” thatdifferentiate to produce the specialized cells found in mammalian tissueand organs.

The term “biocompatible” as used herein refers to materials which havelow toxicity, clinically acceptable levels of foreign body reactions inthe living body, and affinity with living tissues.

The terms “lyophilize” or “freeze drying” refer to the preparation of acomposition in dry form by rapid freezing and dehydration in the frozenstate (sometimes referred to as sublimation). This process may takeplace under vacuum at reduced air pressure resulting in drying at alower temperature than required at full pressure.

The term “residual moisture” as used herein refers to the amount ofmoisture remaining in the dried sample. It is referred to as a percentof the weight of the sample. In one aspect of the invention the fibrinmatrices of the invention have less than 15% residual moisture,preferably less than 10% residual moisture.

The term “cell-bearing” as used herein refers to the capacity of thematrix to allow cells to be maintained within its structure. In oneaspect, the cells are able to invade the pores and channels of thematrix and may undergo proliferation and or differentiation.

The term “implantation” refers to the insertion of a sponge of theinvention into a patient, whereby the implant serves to replace, fullyor partially, tissue that has been damaged, diseased or removed.

The “biologically active” or “bioactive agents” incorporated into thesponge, for example, growth factors, platelet and platelet extracts,angiogenic factors, and the like, are advantageous to, in a non-limitingexample, encourage a more rapid growth or differentiation of the cellswithin the implant, or a more rapid vascularization of the implant. Suchfactors have now been shown to be effectively retained within the spongeand form a source, or depot, of bioactive agent, for sustained release.Other bioactive agents include antibiotics, enzymes, additional plasmaproteins or mixtures thereof.

The “pore size” of a pore within a plasma protein sponge is determinedby using the equation: P=(L×H)^(1/2) wherein, L and H are the averagelength and height of the pores, respectively, as determined bymicroscopic analysis of the various sponges.

“Polysaccharides” as used herein refer to complex carbohydrates made ofmore than one saccharide. Included in the definition are anionicpolysaccharides, including non-modified as well as chemical derivativesthereof, that contains one negatively charged group (e.g., carboxylgroups at pH values above about 4.0) and includes salts thereof, such assodium or potassium salts, alkaline earth metal salts such as calcium ormagnesium salts. Non-limiting examples of anionic polysaccharidesinclude pectin, alginate, galactans, galactomannans, glucomannans andpolyuronic acids.

A “glycosaminoglycan” or “GAG” as used herein refers to a longunbranched polysaccharide molecules found on the cell surface orextracellular matrix. Non-limiting examples of glycosaminoglycan includeheparin, chondroitin sulfate, dextran sulfate, dermatan sulfate, heparansulfate, keratan sulfate, crosslinked or non-crosslinked hyaluronicacid, hexuronyl hexosaminoglycan sulfate, and inositol hexasulfate.Derivatives, salts and mimetics of the above, including low molecularweight heparin are intended to be included in the invention. Withoutwishing to be bound to theory, the presence of the GAGs, in particularheparin aids in immobilizing growth factors, in particular heparinbinding growth factors such as those of the Fibroblast Growth Factor(FGF) family.

The term “cartilage” as used herein, refers to a specialized type ofconnective tissue that contains chondrocytes embedded in anextracellular matrix. The biochemical composition of cartilage differsaccording to type but in general comprises collagen, predominantly typeII collagen along with other minor types, e.g., types IX and XI,proteoglycans, other proteins and water. Several types of cartilage arerecognized in the art, including, for example, hyaline cartilage,articular cartilage, costal cartilage, fibrous cartilage(fibrocartilage), meniscal cartilage, elastic cartilage, auricularcartilage, and yellow cartilage. The production of any type of cartilageis intended to fall within the scope of the invention. The term“chondrocytes” as used herein, refers to cells which are capable ofproducing components of cartilage tissue.

The term “variant” as used herein refers to a polypeptide sequence thatpossesses some modified structural property of the wild type or parentprotein. For example, the variant may be truncated at either the aminoor carboxy terminus- or both termini or may have amino acids deleted,inserted or substituted. It may be antagonistic or agonistic withrespect to normal properties of the native protein. The variant may havesimilar or altered activity as compared to that of the wild typeprotein.

Embodiments of the Invention

The present invention relates to porous, freeze-dried fibrin matricescomprised of plasma proteins substantially devoid of exogenousanti-fibrinolytic agents useful for supporting cell growth. The presentinvention relates to the unexpected finding that a porous, freeze-driedfibrin matrix comprised of plasma proteins substantially devoid ofplasminogen exhibits superior biological characteristics, in particularcell viability and cell proliferation. Plasminogen is a plasma proteinwhich is enzymatically converted to an active serine protease, plasmin,having fibrinolytic activity. This activity results in the rapiddegradation of fibrin in fibrin clots and matrices. Anti-fibrinolyticagents are typically incorporated into fibrin clots and matrices inorder to maintain the integrity of the substrate. The matrices of thepresent invention lack plasminogen thus obviating the need for exogenousanti-fibrinolytic agents, which have been shown to be deleterious tocells and tissue and which may induce adverse reactions in patients. Itis now further disclosed that matrices comprising partially purifiedplasma proteins also obviate the need for exogenous anti-fibrinolyticagents. The compositions and methods of the present invention areeffective in in vivo and in vitro applications including as fullybiocompatible implants for tissue engineering as well as inbiotechnology. The matrices according to the present invention may beused clinically, per se or as cell-bearing implants. They are truethree-dimensional structures capable of providing support and ofmaintaining cell growth and differentiation.

In one aspect, the present invention relates to a freeze-dried fibrinmatrix comprising plasma proteins substantially devoid of exogenousanti-fibrinolytic agents, plasminogen and of organic chelating agents.Substantially devoid of plasminogen refers to plasma proteins comprisingless than about 20% plasmin or plasminogen normally present in plasma,preferably less than about 10% of plasminogen normally present inplasma, more preferably less than about 5% of plasminogen normallypresent in plasma.

The inventors have discovered that a porous freeze-dried fibrin matrixcomprising plasma proteins substantially devoid of exogenousanti-fibrinolytic agents, plasminogen and of organic chelating agentsprovides a superior matrix for clinical and biotechnologicalapplications. In addition to eliminating the need for exogenousanti-fibrinolytic agents and their concomitant detrimental effects, theinventors now show that the fibrin matrix of the present invention issuperior as a scaffold for cell seeding, growth and differentiation andtissue repair and regeneration.

According to one embodiment of the present invention, the fibrin matrixcomprises plasma proteins, the major protein being fibrin. Fibrin isobtained by the interaction of the plasma proteins fibrinogen (Factor I)and thrombin in the presence of calcium ions (Ca⁺²) and Factor XIII oranother fibrin stabilizing factor, to form a fibrin clot. The plasmaproteins utilized in the present invention may be purified from a plasmasource or may be used from a commercially available source, includingnative or recombinant proteins, in the substantial absence of organicchelating agents. Total blood, blood fractions, blood derivative,cryoprecipitate, recombinant proteins, plasma or plasma fractions mayserve as a plasma protein source for the fibrin sponge of the presentinvention. The plasma source may be allogeneic or autologous. Anothersource of the plasma proteins, specifically of fibrinogen, includesfibrinogen variants, including the high molecular weight (HMW), the lowmolecular weight (LMW) and the LMW derivative (LMW′) variants, forexample as disclosed in PCT patent application WO 03/087160.

The plasma proteins are substantially devoid of plasminogen. Plasminogenmay be removed from the plasma by methods known in the art. In onenon-limiting example, the plasminogen is removed from plasma by affinitypurification, Epsilon amino carboxylic acid (EACA) ligands as well aslysine resin have been used to purify plasminogen from whole plasma. PCTpatent application WO 02/095019 discloses a method for specificallyremoving plasminogen and plasmin in the presence of fibrinogen from amixture such as blood or cryoprecipitate. The method requires contactingthe mixture comprising plasminogen with a rigid amino acid, such astranexamic acid, wherein the amino group and carboxylic group are about7 angstroms apart and the rigid amino acid is covalently bound to thesupport via the amino group. PCT patent application WO 95/25748discloses a topical fibrinogen complex essentially free of plasminogenwhereby the plasminogen was removed using a Sepharose®-lysine column.Alternatively, some or all of the plasma proteins may be recombinant andconsequentially devoid of plasminogen, for example as disclosed in PCTpublication WO 99/56797.

The plasma proteins are further substantially devoid of exogenousanti-fibrinolytic agents, which have been shown to be detrimental tocell growth and may induce adverse reactions in patients. Surprisingly,a matrix comprising partially purified plasma proteins also obviates theneed for exogenous anti-fibrinolytic agents.

The fibrin matrix of the invention may be used per se, comprising plasmaproteins substantially devoid of exogenous anti-fibrinolytic agents,plasminogen and of organic chelating agents, for clinical andbiotechnological applications. It may however, further compriseadditives that impart other advantageous biological, physical andmechanical characteristics to the matrix. Copending international patentapplication WO 03/007873 of some of the inventors of the presentinvention discloses a fibrin matrix comprising plasma proteins and atleast one anti-fibrinolytic agent, optionally further comprising agentssuch as polysaccharides, anionic polysaccharides, glycosaminoglycans, orsynthetic polymers added in the preparation to improve certain physical,mechanical and biological properties of the matrix. The incorporation ofat least one such agent was shown to impart superior characteristicsincluding elasticity and regular pore size to the sponge.

In one embodiment, the present invention is related to a porous fibrinmatrix substantially devoid of exogenous anti-fibrinolytic agents,plasminogen and of organic chelating agents further comprising at leastone additive selected from the group consisting of polysaccharides,glycosaminoglycans and synthetic polymers that is useful as a supportfor culturing or growth of cells, both in vitro and in vivo. Theincorporation of at least one additive to the matrix forming materials,results in a sponge having certain advantageous properties includingphysical, mechanical and/or biological properties. The incorporation ofat least one glycosaminoglycan is shown to impart superiorcharacteristics including elasticity to the sponge. The sponges formedare substantially homogeneous having no particles or interruptingsubstructures other than the pores and interconnecting channels.

In one embodiment the additive may be added ab initio, during formationof the clot. In another embodiment the additive may be introduced to thematrix anytime following formation of the sponge. According to variousembodiments of the present invention, the matrix is prepared using atleast one glycosaminoglycan selected from the group consisting ofcrosslinked hyaluronic acid, non-crosslinked hyaluronic acid, heparinand heparin derivatives and mimetics, chondroitin sulfate, dextransulfate, dermatan sulfate, heparan sulfate and keratan sulfate. In oneaspect the glycosaminoglycan is incorporated into the matrix duringinitial formation of the clot. In one embodiment the glycosaminoglycanis hyaluronic acid. The glycosaminoglycan is added to a finalconcentration that imparts suppleness and elasticity to the sponge andprecludes the need for adjusting the moisture content of the finalcomposition. Hyaluronic acid may be crosslinked or non-crosslinked,having a variety of different molecular weights and may originate froman animal source or a recombinant source. According to one embodimentthe concentration of non-crosslinked hyaluronic acid is about 0.005% toabout 0.5% final (v/v) more preferably about 0.05% to about 0.1%. Inanother embodiment the concentration of crosslinked hyaluronic acid isabout 0.001% to about 0.1% and more preferably around 0.05% to about0.09% final concentration. According to one embodiment theglycosaminoglycan is selected from heparin and a derivative thereof.

According to yet another embodiment the present invention may furtherinclude the incorporation of an additional synthetic or natural polymerprior to formation of the clot which may modify certain properties ofthe sponge including physical, mechanical and/or biological properties.These may impart superior characteristics including elasticity, regularpore size and strength to the sponge. Non-limiting examples of naturalpolymers include cellulose, pectin, polyuronic acids, hexuronylhexosaminoglycan sulfate and inositol hexasulfate.

The synthetic polymers useful for the present invention may benon-biodegradable or biodegradable. Examples of non-degradable materialsinclude polytetrafluoroethylene, perfluorinated polymers such asfluorinated ethylene propylene, polypropylene, polyethylene,polyethylene terapthalate, silicone, silicone rubber, polysulfone,polyurethane, non-degradable polycarboxylate, non-degradablepolycarbonate, non-degradable polyester, polyacrylic,polyhydroxymethacrylate, polymethylmethacrylate, polyamide such aspolyesteramide, and copolymers, block copolymers and blends of the abovematerials.

Examples of degradable materials include hydrolyzable polyesters such aspolylactic acid and polyglycolic acid, polyorthoesters, degradablepolycarboxylates, degradable polycarbonates, degradablepolycaprolactones, polyanhydride, and copolymers, block copolymers andblends of the above materials. Other components include surfactantsincluding lecithin.

In one embodiment, the invention provides a heterogeneous spongecomprising particulate matter such as calcium phosphate crystals orother particles. The particulate matter may be incorporated ab initio inorder to provide a matrix having physical or biological characteristicsadvantageous for certain applications.

Bioactive Agents

In one embodiment the matrix of the invention further comprises at leastone bioactive agent, such as a cytokine, a growth factor and theiractivators, platelets, a bioactive peptide etc. Without wishing to bebound by theory, incorporation of such agents into the sponge of thepresent invention provides a slow-release or sustained-releasemechanism. As the matrix degrades in vivo, the bioactive agents arereleased into the surrounding milieu. For example, growth factors,structural proteins or cytokines which enhance the temporal sequence ofwound repair, enhance angiogenesis, alter the rate of proliferation orincrease the metabolic synthesis of extracellular matrix proteins areuseful additives to the matrix of the present invention. The bioactiveproteins of the invention are polypeptides or derivatives or variantsthereof, obtained from natural, synthetic or recombinant sources, whichexhibit the ability to stimulate DNA synthesis and cell division ordifferentiation of a variety of cells, including primary fibroblasts,embryonal stem cells (ESC), adult stem cells, chondrocytes, vascular andcorneal endothelial cells, osteoblasts, myoblasts, smooth muscle andneuronal cells. Representative proteins include bone growth factors(BMPs, IGF) and fibroblast growth factors and their variants, includingFGF2, FGF4, FGF9 and FGF18 for bone and cartilage healing, cartilagegrowth factor genes (CGF, TGF-β) for cartilage healing, nerve growthfactor genes (NGF) and certain FGFs for nerve healing, and generalgrowth factors such as platelet-derived growth factor (PDGF), vascularendothelial growth factor (VEGF), insulin-like growth factor (IGF-1),keratinocyte growth factor (KGF), endothelial derived growth supplement(EDGF), epidermal growth factor (EGF) and other proteins which mayenhance the action of the growth factors including heparin sulfateproteoglycans (HSPGs) their mimetics such as dextran sulfate, sucroseocta sulfate or heparin, and fragments thereof. Other factors shown toact on cells forming bone, cartilage or other connective tissue includeretinoids, growth hormone (GH), and transferrin. Proteins specific forcartilage repair include cartilage growth factor (CGF), FGFs and TGF-β.

Other biologically active agents that may be included into the matrixinclude blood platelets, platelet supernatants or extracts and plateletderived proteins, hormones, analgesics, anti-inflammatory agents,anti-microbials or enzymes. Bioactive agents including platelets andplatelet supernatant or extract promote the proliferation anddifferentiation of skeletal cells including chondrocytes and osteoblastsand of other cell types including but not limited to hepatocytes andendothelial cells. Bioactive agents belonging to the class ofanti-microbial or anti-inflammatory agents may accelerate the healingprocess by minimizing infection and inflammation. Enzymes such aschondroitinase or matrix metalloproteinases (MMPs) may be incorporatedto aid in the degradation of cartilage, thus stimulating release ofcells into the matrix and the surrounding milieu. In one non-limitingexample, the at least one bioactive agent, added ab initio or at anystage following preparation, may be selected to enhance the healingprocess of the injured or diseased tissue.

According to one embodiment of the present invention the at least onebioactive agent is a therapeutic protein selected from the groupconsisting of growth factors and their variants. In one embodiment, thegrowth factor is a fibroblast growth factor (FGF) or bone morphogeneticprotein (BMP) or variant thereof. In another embodiment, the FGF is anFGF or FGF variant having the capacity to induce cartilage and bonerepair and regeneration and or angiogenesis. The growth factors may beincorporated at a wide range of concentrations, depending on theapplication. For certain applications sustained release of a bioactiveagent is preferred. Sustained release of a bioactive agent may depend onseveral factors including growth factor concentration, type ofglycosaminoglycan incorporated and thrombin concentration.

In contrast to the bioabsorbable heteromorphic sponge of the art, thepresent inventors now disclose a freeze-dried homogenous fibrin spongecompromising plasma proteins substantially devoid of plasminogen and oforganic chelating agents further comprising at least one additiveselected from the group consisting of polysaccharides,glycosaminoglycans and synthetic polymers and at least one bioactiveagent providing phasic release of said bioactive agent.

According to various specific embodiments of the present invention theporous fibrin matrix comprising plasma proteins substantially devoid ofantifibrinolytic agents, plasminogen further comprises at least oneglycosaminoglycan and at least one bioactive agent, wherein thebioactive agent is a therapeutic protein belonging to the FGF family ofgrowth factors. In one embodiment a porous fibrin matrix comprisingplasma proteins substantially devoid of plasminogen and of organicchelating agents further comprises hyaluronic acid, heparin and an FGF.In one aspect the hyaluronic acid and the heparin or heparin mimetic areincorporated into the sponge ab initio.

According to one non-limiting example the present invention provides aporous homogenous freeze-dried fibrin matrix comprising plasma proteinssubstantially devoid of plasminogen, substantially devoid of organicchelating agents, further comprising at least one glycosaminoglycan andat least one bioactive agent, wherein the at least one glycosaminoglycanis heparin and the at least one bioactive agent is a therapeutic proteinbelonging to the FGF family of growth factors or a variant thereof. Thissponge provides phasic release of the FGF from the matrix and may bebeneficial in certain therapeutic applications. Optionally, at least onebioactive agent may be added to the cell, either in culture or duringseeding, for example, to enhance a therapeutic effect.

Additionally, cells genetically engineered to express the aforementionedproteins are including in the present invention. According to oneaspect, periosteal cells, mesenchymal stem cells or chondrocytes areused per se or are transfected with cartilage growth factor genesselected from a group including transforming growth factor-β(TGF-β),certain FGFs or CGF for cartilage repair and regeneration; for bonerepair periosteal or other mesenchymal stem cells or osteoblasts areused per se or are transfected with bone growth factor genes selectedfrom a group including bone morphogenetic protein (BMP) family genes orfibroblast growth factor family genes; for nerve repair neural cells andneural support cells are used per se or are transfected with genesselected from a group including nerve growth factor (NGF) gene orspecific FGFs.

Furthermore, specific enzymes maybe admixed with the sponge of theinvention in order to promote degradation of the proteoglycans and/orproteins present in the cartilage. Chondrocytes of the cartilage areembedded in the thick extracellular matrix (ECM) of the joint. Withoutwishing to be bound by theory enzymes known in the art includingcollagenase, trypsin, chymotrypsin, chondroitinase of the various types,are able to degrade the ECM of the surface of the joint, therebyreleasing chondrocytes that are able to invade the sponge of theinvention to promote cartilage regeneration.

The matrix of the invention, in certain embodiments may further includeone or more antiseptics, such as methylene blue, and/or one or moredrugs including antimicrobials such as antibiotics and antiviral agents;chemotherapeutic agents; anti-rejection agents; analgesics and analgesiccombinations; anti-inflammatory agents; adhesion protein such asfibronectin or fragments thereof and hormones such as steroids.

According to one embodiment the at least one bioactive agent isplatelets or platelet supernatant. The platelets may be present in theplasma protein concentrate or may be added exogenously. An exogenoussource of platelets is added during the clot forming process to a finalconcentration of 0.1% to 30% of final sponge volume, more preferably 5%to 25% of final sponge volume. An exogenous source of plateletsupernatant is added during the clot forming process to a finalconcentration of 0.1% to 30% of final sponge volume, more preferably 1%to 15% of final sponge volume.

Applications

The porous homogeneous fibrin matrix of the invention is useful asscaffold for tissue engineering applications. The absence of plasminogenobviates the need for external anti-fibrinolytic agents and thus resultsin a sponge that is fully biocompatible. The optional presence of thebioactive agents and the glycosaminoglycan together provides as anunexpectedly advantageous support for cellular growth in vitro and invivo.

The in vivo uses of the plasma matrix are manifold. The fibrin scaffoldmay be used as an implant per se, for providing mechanical support to adefective or injured site in situ and/or for providing a matrix withinwhich cells from the defective or injured site proliferate anddifferentiate. The cells may be stem cells or progenitor cells or may bespecialized cells such as chondrocytes, osteoblasts, hepatocytes, ormesenchymal, endothelial, epithelial, urothelial, endocrine, neuronal,pancreatic, renal or ocular cell types.

The homogeneous porous fibrin matrix of the present invention can beutilized in reconstructive surgery methods for regenerating and/orrepairing tissue that have been damaged for example by trauma, surgicalprocedures or disease. The present invention provides a matrix for useas an implantable scaffold per se for tissue regeneration. According toone aspect of the invention, the matrix serves as both a physicalsupport and an adhesive substrate for in vivo cell growth. As the cellpopulations grow and the cells function normally, they begin to secretetheir own extracellular matrix (ECM) support. The scaffold polymer isselected to degrade as the need for an artificial support diminishes.

Scaffold applications include the regeneration of tissues such asneuronal, musculoskeletal, cartilaginous, tendonous, hepatic,pancreatic, renal, ocular, arteriovenous, urinary or any other tissueforming solid or hollow organs. Some typical orthopedic applicationsinclude joint resurfacing, meniscus repair, non-union fracture repair,craniofacial reconstruction or repair of an invertebral disc.

The porous fibrin matrix of the invention is useful, inter alia, as anunexpectedly advantageous support for cellular growth. The absence ofexogenous anti-fibrinolytic agents results in a fibrin matrix that isfully compatible with in vitro and in vivo cell growth, proliferationand differentiation. An additional advantage of the fibrin matrix of theinvention is its improved ability to absorb cells and retain them. Theneed to wet or wash the sponge of the invention prior to cell seeding isprecluded by the absence of exogenous anti-fibrinolytic agents. In oneembodiment the matrix of the invention serves as a scaffold for thegrowth, proliferation and/or differentiation of cells including stemcells, progenitor cells or other cell types including chondrocytes,osteoblasts, hepatocytes, mesenchymal, epithelial, urothelial, neuronal,pancreatic, renal or any other cell types which it is desired to culturewithin a three dimensional support.

In a certain embodiment of the present invention cells may be culturedon the matrix for subsequent implantation. Stem cells derived from anytissue or induced to differentiate into a specific tissue type may beutilized. Preferably the cells are derived from autologous tissue. Forexample, for culturing cartilage, chondrocytes or mesenchymal stem cellsmay be seeded on the matrix. In specific embodiments of the invention,chondrocytes or chondrocyte progenitor cells can be seeded on the matrixprior to implantation or at the site of implantation in vivo. The spongeis useful for the delivery of cells in situ to a specific site in thebody, such as dopamine expressing cells to Parkinson's patients.

Additionally, the cell of interest may be engineered to express a geneproduct which would exert a therapeutic effect, for exampleanti-inflammatory peptides or proteins, growth factors havingangiogenic, chemotactic, osteogenic or proliferative effects. Anon-limitative example of genetically engineering cells to enhancehealing is disclosed in U.S. Pat. No. 6,398,816.

According to certain embodiments of the invention, the fibrin matrix isused as a support for chondrocyte growth and as a scaffold for neocartilage formation. However, the plasma matrix of the invention may beused as a surface useful for tissue culture for any suitable cells, suchas mesenchymal cells or other tissue forming cells at different levelsof potency. For example, cells typically referred to as “stem cells” or“mesenchymal stem cells”, are pluripotent, or lineage-uncommitted cells,which are potentially capable of an unlimited number of mitoticdivisions to either renew a line or to produce progeny cells with thecapacity to differentiate into any cell type can be grown on the matrixof the invention. In addition, lineage-committed “progenitor cells” canbe grown on the matrix of the invention. A lineage-committed progenitorcell is generally considered to be incapable of an unlimited number ofmitotic divisions and will eventually differentiate only into a specificcell type. Cell types include chondrocytes, osteoblasts, hepatocytes, ormesenchymal, endothelial, epithelial, urothelial, endocrine, neuronal,pancreatic, renal or ocular cell types.

In yet further embodiments of the invention, the porous homogeneousfibrin matrix can be utilized as a coating of synthetic or otherimplants or medical devices. The matrix of the invention may be appliedto prostheses such as pins or plates by coating or adhering methodsknown to persons skilled in the art. The matrix coating, which iscapable of supporting and facilitating cellular growth, can thus beuseful in providing a favorable environment for the implant orprosthesis.

A person skilled in the art can adjust the procedures exemplified belowin accordance with specific tissue requirements. For example, forcartilage repair the porous, homogeneous freeze-dried fibrin matrix ofthe invention may be used in conjunction with other therapeuticprocedures including chondral shaving, laser or abrasion chondroplasty,and drilling or microfracture techniques.

Preferably, the fibrin sponge is implanted per se, and serves as ascaffold for cellular growth in situ. Alternatively, the matrix isseeded with desired cells, the cells allowed to proliferate and thesponge comprising the cells implanted at a site in need of tissue repairor regeneration. The glycosaminoglycan enriched homogeneous fibrinmatrix, in its dry form, adheres exceptionally well to tissue surfaces.According to one embodiment of the present invention a dry sponge of theinvention, or another type of bioabsorbable matrix, is placed on thearea where tissue regeneration is desired. A second sponge, onto whichparticular cells were cultured, is placed on top of the dry sponge. Thewetted sponge of the invention adheres well to the dry sponge of theinvention or another matrix. During the healing process, the cells fromthe sponge onto which the cells were originally seeded migrate into thematrix adhering directly to the area of tissue regeneration.

In the reconstruction of structural tissues like cartilage and bone,tissue shape is integral to function, requiring the molding of thematrix into three dimensional configuration articles of varyingthickness and shape. Accordingly, the matrix of the invention may beformed to assume a specific shape including a sphere, cube, rod, tube ora sheet. The shape is determined by the shape of a mold, receptacle orsupport which may be made of any inert material and may be in contactwith the matrix on all sides, as for a sphere or cube, or on a limitednumber of sides as for a sheet. The matrix may be shaped in the form ofbody organs or parts and constitute prostheses. Removing portions of thematrix with scissors, a scalpel, a laser beam or any other cuttinginstrument can create any refinements required in the three-dimensionalstructure.

The matrix according to further embodiments of the invention can be usedas a matrix for growing cells or tissue culture in vitro. The matricesof the invention provide a relatively large surface area for cells togrow on and a mechanically improved scaffold for implantation.

The methods for seeding cells on the matrix are manifold. In anon-limiting example, the cells are adsorbed by placing the cells on thesurface of the matrix or absorbed into the matrix by placing the spongein a solution containing cells. The matrix may be seeded with thedesired cells by surface seeding, at a density of about 10⁴ cells percm³, more preferably about 10⁵ cells per cm³.

It will be appreciated that the matrix of the invention can support thegrowth and/or implantation of any type of cartilage or other suitabletissue. Furthermore, although the invention is directed predominantly tomethods for growth and/or implantation of tissue in humans, theinvention may also include methods for growth and/or implantation oftissues in any mammal.

Furthermore, the sponge of the present invention may be used as acomponent of a two-phase or multi-phase material for tissue repair suchas seen in osteochondral defects. In a non-limiting example, one layermay comprise a calcium phosphate material whilst an additional layer maycomprise the sponge of the invention. Gao et al. (Tissue Engin.8:827-837, 2002) describes a method for the repair of osteochondraldefects in rabbit knees using a composite material comprising aninjectable calcium phosphate and a hyaluronic acid sponge.

Method of Matrix Preparation

The present invention provides a method for preparing a poroushomogeneous fibrin matrix. The matrix forming solutions include athrombin solution and a plasma protein solution. As used herein thethrombin solution comprises thrombin in an amount sufficient to cleavefibrinogen and yield a fibrin matrix in the presence of calcium ions(Ca⁺²) ions. The plasma proteins may derive from a commercial,xenogeneic, allogeneic or autologous source and comprise fibrinogen andfactor XIII, substantially devoid of plasminogen and in the substantialabsence of organic chelating agents. The plasma protein solution maycomprise fibrinogen variants such as the high molecular weight or lowmolecular weight variants.

According to one embodiment of the present invention the poroushomogeneous fibrin sponge is prepared by transferring the thrombinsolution into a mold, adding the plasma protein solution; freezing theclotted mixture and lyophilizing. Alternatively, the plasma proteins aremixed with thrombin in the presence of calcium ions under conditionssuitable for achieving clotting; the mixture is cast or mold in a solidsupport prior to achieving clotting; the clotted mixture is frozen andlyophilized. It is to be understood that when incorporated, additivesand bioactive agents are added independently to either of the matrixforming solutions, i.e. the plasma proteins or to the thrombin solution,prior to the formation of the clot or are placed into the mold prior to,concurrently with or following addition of the thrombin.

A method for preparing a porous freeze-dried fibrin matrix formed fromplasma proteins having less than 10% residual moisture and beingsubstantially devoid of exogenous anti-fibrinolytic agents and oforganic chelating agent comprises the following steps:

-   -   providing a thrombin solution and a plasma protein solution        wherein the plasma protein solution is substantially devoid of        exogenous anti-fibrinolytic agents and of organic chelating        agents;    -   introducing the thrombin solution and the plasma protein        solution to a solid receptacle or mold in the presence of        calcium ions; incubating under conditions appropriate to achieve        clotting;    -   freezing the clotted mixture; and    -   lyophilizing the clotted mixture, to obtain a sponge.

According to one embodiment of the present invention the plasma proteinsare partially purified plasma proteins. According to another embodimentof the present invention the plasma proteins are devoid of plasminogen.According to yet another embodiment the plasma protein solutioncomprising less than about 20% of plasminogen normally present in bloodplasma, preferably less than about 10% of the plasminogen normallypresent in plasma and more preferably less than about 5% of theplasminogen normally present in plasma.

According to one embodiment the matrix of the invention may be preparedby sequential introduction of the thrombin solution and plasma proteinsolution into the mold or solid receptacle. Either solution may beintroduced first. According to another embodiment of the presentinvention the thrombin solution and the plasma protein solution aremixed together and subsequently introduced into a mold. The resultingsponges are different in their porosity and cell dispersion.

A method for preparing a porous freeze-dried fibrin matrix having lessthan 10% residual moisture and being substantially devoid of exogenousanti-fibrinolytic agents, and of organic chelating agents furthercomprising at least one additive selected from the group consisting ofpolysaccharides, glycosaminoglycans and synthetic polymers comprises thefollowing steps:

-   -   providing a plasma protein solution substantially devoid of        exogenous anti-fibrinolytic agents and of organic chelating        agents and a thrombin solution and wherein at least one of the        plasma protein solution or the thrombin solution contains at        least one additive selected from the group consisting of        polysaccharides, glycosaminoglycans and synthetic polymers;    -   introducing the thrombin solution and the plasma protein        solution to a solid receptacle or mold;    -   incubating under conditions appropriate to achieve clotting;    -   freezing the clotted mixture;    -   lyophilizing the clotted mixture, to obtain a sponge;

The sponge may further comprise at least one bioactive agent, added abinitio to either the thrombin solution or the plasma protein solution.

According to one embodiment of the present invention the plasma proteinsare partially purified plasma proteins. According to another embodimentof the present invention the plasma proteins are devoid of plasminogen.According to yet another embodiment the plasma protein solutioncomprising less than about 20% of plasminogen normally present in bloodplasma, preferably less than about 10% of the plasminogen normallypresent in plasma and more preferably less than about 5% of theplasminogen normally present in plasma.

According to various embodiments of the present invention plasmaproteins at a concentration of about 20 mg/ml to about 50 mg/ml,substantially devoid of exogenous anti-fibrinolytic agents, plasminogenand of organic chelating agents are mixed with hyaluronic acid and/orheparin and the mixture is added to the thrombin solution in the solidsupport to achieve formation of a clot. The clot is frozen andlyophilized.

According to another embodiment a plasma protein solution comprisingplasma proteins at a concentration of about 20 to about 50 mg/ml,substantially devoid of antifibrinolytic agents and substantially in theabsence of organic chelating agents, comprising hyaluronic acid andheparin bound to FGF are mixed and the mixture added to the thrombinsolution in the solid support to achieve formation of a clot. The clotis frozen and lyophilized.

The final concentration of thrombin may be varied in order to producesponges with distinct biological, physical and mechanical featuresuseful for different applications.

Thrombin concentrations of about 0.5 IU/ml to about 2 IU/ml providesponges with similar properties in terms of cell viability and growth.Other concentrations, as low as 0.15 IU/ml may be useful as well,depending on the application.

In its final form prior to use with cells the sponge is substantiallydry and contains less than 15% residual moisture, more preferably lessthan 10% residual moisture.

Yet another aspect of the present invention provides methods oftreatment and use of the fibrin matrix of the invention for treatinginjured or traumatized tissue, including cartilage and bone defects. Themethod of treatment described herein is advantageous in that it requiresminimal preparation for use by the medical practitioner. The in vivouses of the porous fibrin matrix are manifold. The porous fibrin matrixmay function as a scaffold and may be used as an implant per se, forproviding mechanical support to a defective or injured site in situand/or for providing a matrix within which cells from the defective orinjured site proliferate and differentiate. For example, for cartilagerepair the porous fibrin matrix may be used in conjunction with othertherapeutic procedures including chondral shaving, laser or abrasionchondroplasty, and drilling or microfracture techniques.

The porous fibrin matrix of the invention, being an effective scaffoldsupporting cell growth, may further be utilized in vivo inreconstructive surgery, for example as a matrix for regenerating cellsand tissue including neuronal cells, cardiovascular tissue, urothelialcells and breast tissue. Some typical orthopedic applications includejoint resurfacing, meniscus repair, non-union fracture repair,craniofacial reconstruction, osteochondral defect repair or repair of aninvertebral disc. The fibrin matrix of the invention may serve to treatdefects resulting from disease such as osteoarthritis. The components ofthe matrix may be cast into a mold specifically designed for a distinctlesion or defect. In a non-limiting example, the mold may be prepared bycomputer aided design. In other instances the medical practitioner mayhave to cut or slice the sponge to fit a particular lesion or defect.The matrix of the invention is particularly beneficial for minimallyinvasive surgical techniques such as a mini-arthrotomy or arthroscopyand overcomes the need for fully open joint surgery.

In one embodiment, the porous fibrin matrix may be used as a coating onsynthetic or other implants such as pins and plates, for example, in hipreplacement procedures. Thus, the present invention further providesimplants or medical devices coated with the comprising the porous fibrinmatrix of the invention.

Furthermore, the sponge of the present invention may be used as acomponent of a two-phase or multi-phase material for tissue repair suchas seen in osteochondral defects. In a non-limiting example, one layermay comprise a calcium phosphate material whilst an additional layer maycomprise the sponge of the invention.

The plasma protein solution may be from a commercial source, natural orrecombinant proteins, or may be prepared from plasma. According to oneembodiment of the present invention the plasma protein solution derivesfrom allogeneic plasma. According to another embodiment of the presentinvention, at least one of the components, preferably the plasmaproteins, used for preparing the matrix derives from autologous plasmaor recombinant proteins. According to another embodiment of the presentinvention, all of the plasma components used in preparing the matrix areautologous. The plasma proteins may be isolated by a variety of methods,as known in the art and exemplified herein below, resulting in a fibrinmatrix having substantially similar properties, as measured by poresize, elasticity, compression and cell bearing capabilities. A stablethrombin component may be isolated from autologous plasma, according tomethods known in the art for example those disclosed in U.S. Pat. No.6,274,090 and Haisch et al (Med Biol Eng Comput 38:686-9, 2000).

The resulting fibrin matrix exhibits advantageous properties includingbiocompatibility, pore size compatible with cell invasion andproliferation and ability to be molded or cast into definite shapes.

In one aspect, blood is drawn from a patient in need of tissue repair orregeneration, plasma proteins, are isolated from the autologous plasmaand a matrix prepared thereof. The platelets are optionally isolated andreturned to the plasma. The matrix of the present invention may serve asan implant for use as a scaffold per se or as a cell-bearing scaffoldfor in vivo implantation.

According to one embodiment of the present invention a porous fibrinsponge produced from a fibrinogen solution, wherein the fibrinogensolution is subjected to dialysis with a solution not requiring acomplexing agent, serves as a scaffold for the growth of cells in vitroand in vivo. According to another embodiment the fibrin sponge is formedby the action of a thrombin solution on the dialyzed fibrinogen solutionand subsequently subjected to freeze drying.

While not wishing to be bound by any particular theory the substantialabsence of organic complexing agents may provide the matrix of thepresent invention with properties beneficial to the proliferation andmetabolism of certain cell types. As shown in the examples herein, thematrix of the present invention supports the proliferation of cartilagecells in both in vivo and in vitro systems.

The presence of certain organic complexing agents in a range of 1 to 20mM, necessary for the production of a flexible fibrin web disclosed inU.S. Pat. No. 6,310,267 for wound healing, may in itself have adetrimental effect on the proliferation of certain cell types. The useof a fibrin web for cell growth and proliferation, in vivo or in vitro,has not been disclosed. Nevertheless, it may be possible to culturecertain types of cell types using the webs of the aforementioned patent.

According to one embodiment of the present invention heparin isincorporated into the matrix to a final concentration of about 0.1 ug/mlto about 1 mg/ml. In another embodiment the concentration of heparin isabout 1 ug/ml to about 50 ug/ml. As used herein ug/ml refers to amicrogram per milliliter.

According to another embodiment of the present invention crosslinkedhyaluronic acid is incorporated into the matrix to a final concentrationof about 0.001% to about 0.1%, more preferably about 0.05% to about0.09%.

According to another embodiment of the present invention non-crosslinkedhyaluronic acid is incorporated into the matrix to a final concentrationof about 0.005% to about 0.5%, more preferably about 0.05% to about0.1%.

According to yet another embodiment of the present invention bothheparin and hyaluronic acid are incorporated into the matrix atrespective concentration ranges.

Surprisingly, in view of the known function of heparin as ananti-coagulant, it is now disclosed that the incorporation of heparininto the matrix does not interfere with either the formation of thematrix or the therapeutic benefits of the matrix. Without wishing to bebound by theory, heparin serves primarily to bind FGF or othertherapeutic proteins and creates a depot for sustained release of saidproteins. In addition, low molecular weight fragments of heparinreleased from the matrix may function as anti-inflammatory agents andassist in the healing process of diseased or traumatized tissue (U.S.Pat. Nos. 5,474,987; 5,686,431; 5,908,837).

The following examples are intended to be merely illustrative in natureand to be construed in a non-limitative fashion.

EXAMPLES Example 1 Preparation of a Fibrin Matrix

Although detailed methods are given for the preparation of the plasmaprotein, it is to be understood that other methods of preparing plasmaproteins are known in the art and are useful in the preparation of thematrix of the present invention. A non-limiting example of a protocolfor the preparation of a fibrinogen-enriched solution is given in Sims,et al. (Plastic & Recon. Surg. 101:1580-85, 1998). Any source of plasmaproteins may be used, provided that the plasma proteins are processed tobe substantially devoid of anti-fibrinolytic agents, plasminogen and oforganic chelating agents Examples of plasma protein preparation methodsare given in examples 2 and 3, hereinbelow.

Materials and Methods:

Source of plasma proteins e.g. Plasminogen-free fibrinogen (Omrix, Ill.)approximately 50-65 mg/ml stock solution.

Calcium Chloride 5 mM

Thrombin (1000 International Units/ml, Omrix, Ill.)

Optional: Hyaluronic acid; crosslinked (Hylan (Synvisc), approx. MW6×10⁶, Genzyme, US) or non-crosslinked (approx. MW 8×10⁵, MTF, US;approx. MW 3.6×10⁶, BTG, IL)

The concentration of thrombin determines the reaction time for thepolymerization of the fibrin monomers and contributes to the pore sizeand fiber thickness of the final sponge. A concentration of about 0.15IU to about 15 IU thrombin/mg plasma proteins yielded a sponge with goodphysical and biological properties. The concentrations of about 1 toabout 1.5 IU thrombin/mg plasma proteins was chosen because it gave afast reaction but allowed adequate time for pouring the two solutions(plasma protein and thrombin) before the reaction completes. It shouldbe noted that other concentrations are acceptable for obtaining a matrixwith substantially similar properties. For convenience, as used herein1.5 IU thrombin/mg total protein is the equivalent of about 30 IUthrombin/ml.

The plasma protein solution and the thrombin solution were mixedtogether in a ratio of approximately 2:1 (for example 210 μl plasmaprotein and 90 μl thrombin solution) in the following order: A 48 wellELISA plate was coated with 90 μl of thrombin solution, and the plasmaprotein solution was added. Alternatively, a 96 well ELISA plate wasused and about 19.5 ul thrombin solution was added to the wells followedby the addition of about 45.5 ul plasma protein; or for a slightlythicker matrix about 24 ul thrombin solution and 51 ul plasma protein.The mixture was incubated at room temperature (−25° C.) for about 10minutes or until the clot formed, followed by freezing at about −60° C.to about −90° C. from about 30 minutes to several days. The 48 well sizesponges were lyophilized for about 5 hours while the 96 well platesponges were lyophilized for about 4 hours. The 96 well plate yieldssponges of about 5 mm diameter and the 48 well plate yields sponges ofabout 10 mm (1 cm) diameter (about 0.8 cm²).

A 35 mm diameter sponge has been prepared for the repair of largerdefects, such as those that may develop in osteoarthritis. A 35 mmsponge (about 9.5 cm², about 2 to about 2.5 cm³) was prepared by mixing2 ml plasma protein solution with 1 ml thrombin, casting into anappropriate mold, such as a 35 mm petri dish or a 6-well cell cultureplate, frozen and lyophilized at −40° C. for about 12 hours. The fibrinsponges were prepared under aseptic conditions. It is to be noted thatthe solutions may be cast into a mold of any desired shape. The spongethat resulted was a fleece-like matrix.

According to other embodiments of the present invention the matrix isprepared with certain additives including polysaccharides,glycosaminoglycans and synthetic polymers. Biological, mechanical andphysical parameters were shown to be controlled by incorporating thoseadditives. All additives were filtered (0.2 μm) and were added to theplasma protein solution. When hyaluronic acid was incorporated in thematrix, the plasma protein solution and hyaluronic acid solution wereincubated together before casting. A non-limiting sample list of theadditives and concentrations tested are shown in the Table 1 below:

TABLE 1 Additive % final concentration Glycerol 0.005; 0.01; 0.05; 0.1;0.5; 1 Crosslinked (X-linked) HA 0.0024; 0.012; 0.024; 0.05; 0.10; 0.5Non-X-linked HA 0.002; 0.02; 0.05; 0.07; 0.08; 0.09; 0.1; 0.11; 0.13Heparin 0.05; 0.1; 0.5; 1.0; 2.5; 10 ug/ml final Heparin + CrosslinkedHA Combinations of above Heparin + Non-X-linked HA Combinations of aboveGlycerol + HA Combinations of above Note: Non-X-linked HA refers tonon-crosslinked hyaluronic acid.

A therapeutic protein, FGF (about 1 to about 10 ug/0.2 cm² sponge) wasadded either to the plasma protein solution or was mixed with heparinand then added to either the plasma protein or thrombin solutions.Experiments have been performed to determine the optimal concentrationof the additives in terms of matrix flexibility, elasticity, pore size,sustained release of bioactive agents and cell growth capacity. Theadditives impart beneficial properties, including surface, mechanicaland/or biological properties, to the sponge during its preparation.Optimization was carried out regarding the concentration of thebioactive agents as well. In one embodiment the bioactive agents includegrowth factors, platelet supernatant, native platelets, plateletmembranes and other materials. According to one embodiment the presentinvention provides a matrix comprising heparin or a derivarive thereofand hyaluronic acid further comprising FGF or FGF variant. Examples arepresented herein below.

Example 2 Isolation of Partially Purified Plasma Proteins from WholePlasma

Plasma protein may be prepared from different sources such as freshplasma, fresh frozen plasma, recombinant proteins and xenogeneic,allogeneic or autologous blood. The fresh frozen plasma was receivedfrom the blood bank (Tel-Hashomer, Israel). The plasma (220 ml) wasthawed in a 4° C. incubator over night, followed by centrifugation at 4°C. at approximately 1900 g for 30 min. The pellet was resuspended in 2.5ml PBS with gentle rolling until a homogenized solution was seen. Thetotal protein concentration may be estimated by Bradford assay andSDS-PAGE (comparing to a standard). Exemplary samples were found to beabout 42 mg total protein/ml to about 50 mg total protein/ml. The plasmamay further be treated to remove plasminogen, using methods known in theart. Non-limiting examples of methods useful for removing plasminogenfrom blood or blood derivates such as plasma or a cryoprecipitate aredisclosed in PCT patent publications WO 02/095019 and WO 95/25748.

It is to be understood that the plasma protein source may be xenogeneic,allogeneic or autologous blood. Preferably, the plasma protein source isallogeneic or autologous. A non-limiting method for the isolation of aplatelet-enriched plasma is disclosed in U.S. Pat. No. 6,475,175.

Another embodiment of the present invention provides a plasma proteinsponge incorporating at least one additive and blood platelets orplatelet supernatant. Sponges comprising 0.024% or 0.08% finalconcentration hyaluronic acid and 1% or 10% final concentration plateletreleased supernatant or whole platelets were prepared. Plateletsupernatant was made by exposing isolated platelets (obtained from theIsrael blood bank) to thrombin as described (Gruber et al., Clin OralImplants Res 13:529-535, 2002), collecting the supernatant and adding itto the plasma protein solution prior to sponge formation. Spongescomprising platelets were prepared by adding platelets directly to theplasma proteins in the following manner: 73 ul platelets and additive(hyaluronic acid to 0.024% or 0.08% final concentration) was added toplasma proteins (30 mg total protein/ml) and the solution brought to 210ul final volume. The sponge was made as described hereinabove utilizingpartially purified plasma proteins.

Example 3 Extraction of Plasma Protein Fractions from Allogeneic orAutologous Blood Materials:

1) Sodium citrate, 3.8% or any other pharmaceutically acceptableanti-coagulant2) Ammonium sulfate (NH₄)₂SO₄, saturated (500 g/l)3) Ammonium sulfate (NH₄)₂SO₄, 25%4) Phosphate-EDTA buffer: 50 mM phosphate, 10 mM EDTA, pH 6.65) Tris-NaCl buffer: 50 mM Tris, 150 mM NaCl, pH 7.46) Ethanol, absolute 4° C.7) Whole blood (Israel Blood Bank, Tel Hashomer Hospital or frompatient)

Methods:

This method may be used to produce plasma proteins that may be treatedfor removal of plasminogen by methods known in the art, includingaffinity chromatography. The plasma proteins are isolated according tostandard methods. To one 450 ml bag of blood from the blood bank,containing sodium citrate, 50 ml of a 3.8% sodium citrate solution wasadded and the solution was mixed gently.

The blood-sodium citrate was centrifuged at 2,100 g for 20 min. Thesupernatant plasma was collected re-centrifuged at 5000 g for 15 min. at4° C. The supernatant plasma was put on ice, and saturated ammoniumsulfate solution was added at a ratio of one volume ammonium sulfate to3 volumes of supernatant (1:3 volume ratio). The solution was kept at 4°C. for 1.5 hrs with occasional mild shaking (magnetic stirring is notallowed). The supernatant plasma was centrifuged at 5000 g for 15 min at4° C. The supernatant was discarded and each pellet washed with 10 ml of25% ammonium sulfate solution (pellet not dissolved). Each pellet wasdissolved in 6-7 ml of the phosphate-EDTA buffer. A sample, typically100 μl of the solution, was kept for SDS-PAGE and clotting analyses. Thedissolved pellets were pooled and the ammonium sulfate precipitation wasrepeated by adding saturated ammonium sulfate to the plasma sample toachieve a 1:3 volume ratio (Typically, 25 ml ammonium sulfate to 75 mlplasma). The solution was kept at 4° C. for 1.5 hrs with occasional mildshaking, and centrifuged at 5000 g for 15 min. The supernatant wasdiscarded and the pellets were dissolved in a volume of Tris-NaCl bufferthat was equal to or less than the volume of phosphate-EDTA buffer usedabove. A typical total amount was about 45 ml.

The sample may be dialyzed (SnakeSkin™ dialysis tubes, 3.5 kD cutoff,Pierce) for 3-4 hours or overnight at 4° C. in 1.5 liters of Tris-NaClbuffer. The sample was centrifuged in high-speed resistant tubes at21,000 g for 15 min at 4° C. to remove any insoluble material. Thesupernatant was collected and kept on ice.

The supernatant was ethanol precipitated by adding ethanol to a finalconcentration of 7% and kept on ice for 30 min. The solution wascentrifuged at 5000 g for 15 min, the supernatant discarded and thepellet dissolved in the same volume (typically about 45 ml) Tris-NaClbuffer. The solution was dialyzed overnight at 4° C. in 1.5 liter ofTris-NaCl Buffer. The dialyzed solution was centrifuged at 21,000, at 4°C. for 15 min, to eliminate any non-dissolved material.

Protein concentrations were determined using the standard Bradfordmethod. The protein yields ranged from 0.2 to 0.6 mg per ml of fullblood, with typical results of about 0.4 to 0.5 mg/ml. Clot formationability was determined by adding 30 μl thrombin (100 IU/ml; Omrix) to 70μl plasma product (10 mg/ml), clotting should occur within 30 sec.

Protein purity was determined by electrophoretic analysis of 50 μg ofthe sample on a 5% SDS-polyacrylamide gel and staining using Coommassieblue. The remainder of the supernatant was collected, frozen andlyophilized until dry, 48 hours.

Example 4 Presence of Plasmin and Plasminogen in Plasma Protein Sample

The plasma proteins substantially devoid of plasminogen typicallycomprised about 9 to about 10 ug plasmin and plasminogen per eachmilliliter of total protein, as identified by a polyclonal antibody thatdetects both the plasminogen and plasmin. Human plasma typicallycomprises approximately 200 mg plasminogen per liter or about 200 ug/ml.

This experiment was designed to determine the concentration of plasminthat could be tolerated in a plasma protein clot. The same experimentaldesign is used for testing the tolerance for plasminogen. Plasminogen isthe precursor of the active serine protease plasmin, which is capable ofdegrading fibrin.

Two concentrations of plasmin (ICN Biomedical, 194198, stock 20 mg/ml),0.09 mg/ml, 0.045 mg/ml were added to the plasma proteins substantiallydevoid of plasminogen (Omrix), prior to casting of the solutions. Theplasmin concentration of 0.09 mg/ml represents about a ten-fold greaterplasmin concentration than the total plasmin and plasminogenconcentration present in the commercially available plasma proteins. Theplasmin concentration of 0.045 mg/ml represents about a five-foldgreater plasmin concentration. The plasma protein solution for thesponge comprising 0.09 mg/ml plasmin was prepared by mixing 281 ulplasma proteins (64 mg/ml), 67.5 ul hyaluronic acid, 2.7 ul plasmin and251.3 saline. The plasma protein solution for the sponge comprising the0.045 mg/ml plasmin was prepared by mixing 281 ul plasma proteins, 67.5ul hyaluronic acid, 1.35 ul plasmin and 250 ul saline. A control withoutthe addition of plasmin was prepared. Five sponges were prepared fromeach solution by adding 43 ul thrombin to a well (1 IU thrombin/mgplasma proteins) and 87 ul of the plasma protein solution and themixture allowed to set at room temperature.

Neither of the mixtures comprising plasmin formed a clot, while theplasmin-free control formed a clot within minutes and a freeze driedsponge was formed following freezing and lyophilization. This indicatesthat the plasma proteins may tolerate less than 45 ug/ml plasmin or lessthan about 22.5% of the plasminogen and plasmin normally present inplasma.

Example 5 Matrix Morphology and Mechanical Properties

In general, matrices for tissue engineering are characterized accordingto several criteria, including chemical nature, homogeneity, porosity,adhesion, biocompatibility and elasticity, amongst others (Hunziker,Osteoart. Cart., 10:432-465, 2002). Table II of the aforementionedreference lists several of the properties and the biological basis ofthese properties.

Several of the aforementioned properties are measured for the matrix ofthe invention. Porosity, important for cell migration and adhesion isdetermined by geometrical measurements using the light microscope bysectioning the matrix into thick specimens. Specimens are mounted onslides and are stained by hematoxylin/eosin. An optical micrometermeasured the pore size and the distance between neighboring pores.

Scanning Electron Microscope (SEM) Analysis is performed in order toanalyze homogeneity and ultra structure of the matrix. The thickness ofthe fibrin fibers is measured in this way, as well.

Moisture and residual moisture are measured using standard tests, knownin the art. In its final form prior to use with cells the sponge issubstantially dry and contains less than 15% residual moisture, morepreferably less than 10% residual moisture.

Mechanical property measurements are performed, for example, using aChatillon TCD200 machine with a digital force gauge DF12. Each plasmaprotein sponge is 2.5 cm long, 0.5 cm wide; and is fully lyophilized.Deformation represents the elasticity of the sponge, i.e. the amount ofpull as measured in millimeters (mm) that may be exerted until thesponge tears. Force is calculated in kiloPascal (kPa) and represents theamount of energy required to tear the sponge strips. The thickness ofthe sponge is taken into consideration when making the calculation.

Example 6 Cell Seeding on the Matrix

Different methods of seeding cells onto the sponge may be used.Important to seeding is cell adherence, migratory capacity andproliferation of cells within the matrix. Cells may be suspended inmedium, PBS, or any biocompatible buffer alone or in the presence ofbioactive agents. Cells may be seeded by placing a drop of liquidcontaining cells on the sponge and allowing the cells to adsorb into thesponge. Alternatively, the cells in the liquid may be absorbed into thesponge by placing the sponge in a container holding a suspension ofcells. Other methods including spray seeding have also been shown to beeffective.

One particular advantage of the present invention is the high level ofcell viability and excellent cell distribution following cell seedingdirectly on a dry sponge. Often a matrix comprising an exogenousanti-fibrinolytic agent such as tranexamic acid exhibits lower cellviability following seeding. The cells seem to recover but the exogenousanti-fibrinolytic agents may be detrimental to initial cell growth. Whensuch a sponge is washed and some or all of the tranexamic acid isremoved cell proliferation is improved. It is also noted that many cellssettle at the periphery of the matrix following on a wet sponge whilethere is a better cell distribution following seeding on a dry sponge.

Materials and Methods:

Sponges comprising different concentrations of plasma proteins andthrombin were tested. Sponges comprising 10 mg/ml, 15, 16.5 mg/ml, 18mg/ml, 20 mg/ml, 22 mg/ml, 25 mg/ml, 30 mg/ml and varying concentrationsof hyaluronic acid (from about 0.05% to about 1.1%) and either 1, 1.5 or2 IU thrombin/mg proteins. A total of about 5×10⁵ to about 5×10⁶chondrocyteswere seeded on 1 cm diameter sponges and allowed to incubatefor three days. Different volumes of growth media were added and thecell-embedded matrix allowed to incubate. It is to be understood thatthe sponge of may be of varying sizes, shapes and thickness.

Following a three-day, 1 week and three week incubation for the seededsponges, some of the sponges were collagenase degraded and cells countedfollowing trypan blue staining. Cell proliferation is determined asdescribed in Example 8, below.

Samples of the cell-bearing sponges or matrices, were paraffin-embeddedand sections prepared using a microtome. The histological sections arefurther stained using different biological stains including hematoxylinand eosin (H&E), toluidine blue and fast red, Masson's trichrome stainand others. All sponges exhibited similar cell distribution, with livecells present throughout all layers of the sponge.

Examples of cell growth in the fibrin sponge of the invention are shownin FIGS. 1A and 1B and FIG. 2. Each sponge was seeded with 5×10⁶ porcineor human chondrocytes in 30 microliter volume, allowed to incubate onehour and fresh media was added. After three days the sponges weredegraded in collagenase and the number of live cells was counted afterstaining with trypan blue. FIG. 1A shows the increased viability ofporcine chondrocytes following a three day incubation seeded on matriceswith (speckled) and without plasminogen (solid). After three days, morethan 50% of the cells remained viable as compared to about 20% of thecells seeded on the standard matrix prepared from plasma proteinscomprising tranexamic acid. FIG. 1B shows the viability of humanchondrocytes seeded on matrices with (speckled) and without plasminogen(solid-dry) following a three-day incubation. The plasminogen freesponges showed superior cell viability when seeded with humanchondrocytes when compared to the sponges comprising tranexamic acid.FIG. 1C shows cell viability after three days on three different spongecompositions. All sponges comprised plasma proteins substantially devoidof plasminogen and were seeded with about 4×10⁶ human chondrocytes. Thespeckled bar represents viability on a 20 mg plasma protein/ml sponge,without additive present. The solid black bar represents cell viabilityon a sponge comprising 20 mg/ml and 0.075% hyaluronic acid. Thecheckered bar represents cell viability on a sponge comprising 18 mg/mland 0.75% hyaluronic acid. It can be seen that all three sponges providea good scaffold for cell seeding. FIGS. 1D and 1E show photographs ofone centimeter (1 cm) diameter dry sponges and as cell-bearing implants,respectively.

FIG. 2A-2C show histological cross sections through the center of amatrix comprising human chondrocytes following 1-week incubation. Thefibrin sponge was made of commercial fibrinogen substantially devoid ofplasminogen (Omrix, 20 mg/ml) comprising 0.05% hyaluronic acid and 1×10⁶human cells. Note the infiltration of the chondrocytes into the sponge.FIGS. 2A, 2B and 2C show 40×, 100× and 400× magnifications,respectively.

Example 7 In Vitro Degradation Assay

The assay was carried out to determine the rate of degradation of thesponge of the invention. Differences in the degradation rate can be seenbetween the sponge of the invention and a standard sponge comprisingfibrinogen and an exogenous anti-fibrinolytic such as tranexamic acid.

The assay was performed in the following manner: three different typesof sponges were prepared, each having the same fibrinogen concentration,the same thrombin concentration (1.5 U/mg protein) and the samehyaluronic acid concentration. The differences were the source offibrinogen and hyaluronic acid.

A fibrin sponge comprising 10% tranexamic acid, fibrinogen (Omrix, 27mg/ml), crosslinked hyaluronic acid (Syvisc, 0.08%).

A fibrin sponge prepared from fibrinogen substantially devoid ofplasminogen (Omrix, 27 mg/ml) crosslinked hyaluronic acid (Synvisc,0.08%).

A Fibrin sponge prepared from fibrinogen substantially devoid ofplasminogen (Omrix, 27 mg/ml) non-crosslinked hyaluronic acid (BTG,0.08%).

The experiment was performed as follows: Five sponges prepared in 96well plates were placed in 48 well plates and 750 ul of 10M urea wasadded to cover the sponges.

Samples of 20 ul were collected from each well at the following points:1, 2, 3, 4, 5, 8 minutes, 10 minutes, 30 minutes, 1 hrs. Protein fromeach sample was measured in a standard Bradford assay. The results arepresented in FIG. 3A.

The sponge (

) comprising standard fibrinogen and 10% tranexamic acid underwent rapiddegradation as measured by protein (mg/ml) detected in the supernatantand could not be seen after 10 minutes, whereas the sponge comprisingthe plasma proteins substantially devoid of plasminogen remained stable.

In a similar experiment, the sponges were degraded with collagenase. Thetest sponges were incubated in 400 μl of collagenase (1.7 mg/ml) diluted1:10 in DMEM without FBS at 37° C. until completely dissolved. Atdifferent time points, samples were collected and examined for proteinconcentration by Bradford assay. The results are presented in FIG. 3B.The sponge comprising 10% tranexamic acid and cross linked hyaluronicacid (X link) degraded much faster than the sponges comprising thefibrinogen substantially devoid of plasminogen (wo plm) comprisingeither cross linked or non-cross linked hyaluronic acid (wo plm×link, woplm non×link, respectively).

Example 8 Release of Bioactive Agents from the Matrix

For certain applications, sustained release of a bioactive agent such asa growth factor may be desirable. The incorporation and release ofgrowth factors from the matrix of the invention was assessed in vitroand may be assessed in vivo using radiolabeled or tagged growth factors,for example fluorescent-labeled, alkaline phosphatase labeled orhorseradish peroxidase-labeled growth factor. The fraction and rate ofreleased agent is measured by following the radioactivity, fluorescence,enzymatic activity or other attributes of the tag. Similarly, release ofenzymes from the matrix is determined by analyzing enzymatic activityinto the microenvironment in an in vitro or in vivo assay. Specifically,the release of an FGF from the matrix of the invention was performed asdescribed herein.

The rate of growth factor release was determined from sponges preparedin two alternate methods. In one instance FGF2 was adsorbed to heparinand the combined product was added to the plasma protein solution. Inthe second instance, each component was added separately to theindividual solutions: heparin was added to the plasma protein solutionwhile FGF2 was added to the thrombin solution. Sponges were cast fromboth mixtures and FGF2 release was determined in an FDCP (FactorDependent Cell-Paterson) assay, vide supra.

Materials and Methods:

Plasma proteins (approximately 20-65 fibrinogen mg/ml; Omrix,plasminogen-free).

Non-cross linked hyaluronic acid (MTF or BTG),

Heparin (Sigma, MW 6,000)

FGF2 (ProChon) 2.5 ug per 75 ul sponge

Fibrin sponges substantially devoid of plasminogen and of organicchelating agents were prepared using the method described in Example 1with the following modification: the plasma protein solution comprisednon-crosslinked hyaluronic acid to a final concentration of 0.08%.

The first set of sponges was prepared by mixing heparin solutions withFGF2 and adding the mixture to the plasma protein solution.

The second set of sponges was prepared by adding heparin to the plasmaprotein solution to a final concentration of 0.1, 0.5 or 2.5 ug/ml. FGF2was added to the thrombin solution to bring the final concentration to2.5 ug/sponge. The sponges were cast as described above. FGF2 releasewas determined in a FDCP assay as described below.

FDCP Assay: The FDCP cell line is a murine immortalized, interleukin3-dependent cell line of myelocytic bone marrow origin that does notexpress endogenous FGF Receptors (FGFR). Upon transfection with FGFRcDNA, the FDCP cell line exhibited a dose-dependent proliferativeresponse to FGF that can replace the dependence on IL-3. FGFRtransfected FDCP cells can therefore been used to screen for FGFRsignaling. FDCP cells response to various ligands is quantitated by acell proliferation assay with XTT reagent (Cell Proliferation Kit,Biological Industries Co.). The method is based on the capability ofmitochondrial enzymes to reduce tetrazolium salts into a colorigeniccompound, which can be quantitated and is indicative of cell viability.

Specifically, FDCP cells stably expressing the FGFR1 (FDCP-FGFR1) weregrown in “full medium” (Iscove's Medium containing 2 ml glutamine, 10%FCS, 100 ug/ml penicillin, ML/ml streptomycin) supplemented with 5 ug/mlheparin. Cells were split every 3 days and kept in culture no more thanone month. One day prior to the experiment the cells were split. Beforethe experiment the cells were washed 3 times (1000 rpm, 6 min) with fullmedium. The cells were resuspended and counted with Trypan Blue. Twentythousand (2×10⁴) cells were added to each well of 96-well plate in 50 μlfull medium with or without heparin. Conditioned medium from the spongescontaining FGF or FGF complexed with the various glycosaminoglycans wasadded in an additional volume of 50 μl full medium to bring the finalvolume to 100 μl. The plate was incubated for 48 hours at 37° C. To testcell proliferation, 100 μl of PMS reagent was added to 5 ml of XTTreagent and mixed well (according to manufacture protocol). 50 μl of thelatter solution were aliquoted into each well, and the plates incubatedat 37° C. for 4 hours and the color developed was read by aspectro-ELISA reader at A_(490nm).

FIGS. 4A and 4B show the results of this assay for a sponge of theinvention made of commercial plasma proteins substantially devoid ofplasminogen (Omrix, final 20 mg/ml) comprising 0.08% non-crosslinkedhyaluronic acid, either by adding heparin and FGF2 variant that havebeen premixed (mix) to the plasma protein component of the sponge abinitio or by adding the heparin to the plasma protein solution and theFGF2 variant to the thrombin solution (sep), followed by mixing andcasting the clot. The sponges comprised either 0.5, 1.5 or 2.5 ug/mlheparin and 1 ug total FGF2 variant. Supernatant was tested aftervarious days and results for proliferation recorded. FIG. 4A shows therelease of FGF2 variant from a sponge comprising both heparin and FGF2vafter 1, 3 and 5 days. FIG. 4B shows the percent of total release after5-37 days. The release profile of FGF is dependent on the concentrationof heparin in the sponge. Without wishing to be bound to a particulartheory, the heparin may serve to stabilize the released FGF.

FIG. 4C shows the release profile over 5 days of an FGF variant from asponge comprising heparin and either commercial fibrinogen andtranexamic acid or plasma proteins, substantially devoid ofantifibrinolytic agents. The results show a good release profile forboth compositions.

Example 9 Chondrocyte Isolation and Culturing Reagents:

-   -   Collagenase Type 2; Worthington Biochemical Corp. (Cat. #: 4147)    -   Stock solution: 1700 units/ml in medium (in MEM)    -   Minimal Essential Medium (MEM) Gibco BRL (cat: 21090-022)    -   Fetal Bovine Serum (FBS); Gibco BRL (cat: 16000-044)    -   L-Glutamine Solution; Gibco BRL (cat: 25030-024)    -   Complete medium: Minimal Essential Medium (MEM) supplemented        with 10% fetal calf serum (FCS), 2 mM L-Glutamine and 100 U/ml        penicillin, and 100 μg/ml streptomycin

Preparation of Implants for Articular Cartilage

The sponge of the present invention may be used as a cell bearingscaffold for tissue repair and regeneration. In one aspect, the cellsare cultured on the sponge in vitro, prior to implantation. In anotheraspect, the sponge is seeded with cells immediately before implantationand the cells allowed to proliferate in vivo.

Cartilage biopsies from fresh pig cartilage were sectioned into smallpieces, approximately of 3-4 mm thick, washed aseptically with PBS andplaced in a new tube containing 3 ml MEM medium. The cartilage may beobtained from any vertebrate species, and is preferably allogeneic orautologous.

Collagenase type II was diluted 1:5 and 1 ml was added to the cartilagepieces and the mixture was shaken gently in a 37° C. incubator overnight. When most of the sample was digested, the suspension was pouredthrough sterile gauze to remove matrix debris and undigested material.The filtrate was centrifuged and washed twice to remove residual enzyme.

The number of cells was determined by a hemocytometer and viability wasdetermined by Trypan blue exclusion. The cells were plated in 150 cm²tissue culture flasks in 30 ml of culture medium at a concentration of5×10⁶ cells/ml. Flasks were placed in a 37° C. incubator at 5% CO₂atmosphere and 95% humidity. The culture medium was changed every threeto four days. The cells adhere and become confluent following one weekincubation.

At confluence, the cell medium was removed and 3 ml of a trypsin-EDTAsolution were added. Thirty ml MEM+ FBS was added, the solution wascentrifuged at 800 g for 10 minutes. The supernatant was removed, thepellet dispersed and the cells were counted. To create a cell-bearingmatrix, 10²-10⁶ cells were seeded on a fibrin scaffold of 9 mm indiameter and a thickness of 2 mm (approximately 0.2 cm³). The matriceswere placed in a 37° C. incubator for 1 hour and 1 ml of fresh mediumwas added to each. The medium was replaced with fresh medium and everyfew days the matrices were taken to cell proliferation anddifferentiation analysis.

Furthermore, the cell population grown on the above matrices expressesseveral of the chondrocyte differentiation markers. One of severalphenotypes expressed during chondrocyte differentiation isglycosaminoglycan (GAG) production. The presence of GAGs may beidentified in histological staining using Alcian blue and quantitatedusing the DMB (3,3′-dimethoxybenzidine dihydrochloride) Dye method.Cartilage extracellular matrix may also be identified by staining withtoluidine blue and fast red.

Example 10 Cell Proliferation Assay

Proliferation of the cartilage cells on the matrix of the invention wasquantitated by one of two methods, CyQUANT® (Molecular Probes) or XTTreagent (Biological Industries, Co.). The fibrin matrix was dissolved incollagenase or other enzymes and the cells collected by centrifugationand subjected to analysis according to manufacturer's protocols.

In one experiment, human articular chondrocytes (10⁴-10⁶ cells/30-100ul) were grown on matrices substantially devoid of exogenousanti-fibrinolytic agents in microwell plates. The cells were grownovernight in MEM, 34 U collagenase was added and the cells or cellswithin sponge incubated for four hours. XTT reagent was added for 3-4hours and the plates were read in an ELISA reader at A490 mm. Theresults show that the proliferation rate of the cells was not impairedby the presence of the sponge nor by the addition of the collagenase.FIGS. 5A-5D show porcine chondrocytes (0.5×10⁶ cells in 30 ul) that havebeen cultured (6 days) on a fibrin sponge made from pooled human plasma(30 mg/ml) comprising 0.024% Hylan and 1 ug FGF variant. FIGS. 5A and 5Bshow hematoxylin and eosin (H&E) staining (×100 magnification). FIG. 5Cshows a 400× magnification of a sponge section stained with Masson'sstain. Note the staining for cells and intracellular matrix surroundingthe cells. FIG. 5D shows a ×200 magnification section of sponge stainedwith Masson's stain. Note the cells present within many of the pores.

Example 11 Seeding and Growth of Cells and Cell Lines on the FibrinMatrix

In order to determine the capacity of the plasma protein matrix tosupport cell growth several different cell types and cell lines wereseeded and allowed to grow. Specifically, a primary rat liverhepatocytes were cultured on the matrix. One cm diameter spongescomprising plasma proteins substantially devoid of plasminogen (20 mgprotein/ml, Omrix), 0.075% hyaluronic acid and 1 IU thrombin/ml wereprepared. Approximately 6.6×10⁵ primary hepatocytes were seeded on thesponges in HDM (hormonally defined medium) without serum and allowed toincubate for three days at which histological samples were made andstained with H&E. FIG. 6A shows a representative section of a spongecomprising the hepatocytes. Note the good dispersion of the cellsthroughout the matrix and the presence of typical cells maintainingtheir hepatic characteristics.

Two cell lines were tested for viability and growth within the sponges,the L8 rat skeletal muscle cell line and the CHO Chinese hamster ovarycell line. Two million cells were seeded on each matrix and allowed toincubate for three days. The CHO cell line was cultured in Iscove'smedium, the L8 line was cultures in DMEM. Histological sections weremade and stained with H&E. FIGS. 6B and 6C show sponge sections with theCHO and L8 cells, respectively. In addition, the CHO and L8 cells wereremoved from several of the sponges and counted using Trypan blue. TheL8 cells exhibited 57-67% viability while the CHO cells exhibited morethan 85% viability. Both figures show good cell distribution and cellviability. The matrix of the invention provides a superior scaffold fortissue engineering and regeneration.

Example 12 Ectopic Cartilage Formation in Nude Mice

The assay was designed to determine the ability of isolated chondrocytesto create neocartilage in an ectopic site, and to determine the qualityof this cartilage compared to natural cartilage.

Human and porcine chondrocytes seeded on a matrices of the inventionwere used to induce ectopic cartilage on the backs of nude mice

Treatment arms: The study groups included different amounts of cellsseeded onto the fibrin matrix substantially devoid of plasminogen.Either 10e5 (10̂5) or 10e6 (10̂6) human or porcine chondrocytes wereseeded onto a fibrin sponge from a 96 well plate (˜65 ul). The controlgroup consisted of matrices implanted without cells.

Preparation of fibrin matrices: The method for sponge preparationconsists of mixing a plasminogen free fibrinogen solution (Omrix), witha thrombin solution (Omrix) in the presence of non-crosslinkedhyaluronic acid (BTG), resulting in final concentration of 20 mgfibrinogen/ml, 0.5 IU thrombin/mg fibrinogen and 0.08% hyaluronic acid.The solutions were added to a mold (96 well plate, volume of 650) whereclotting took place at room temperature. The clot was rapidly frozen at−70° C. followed by lyophilization resulting in an implant (matrix,sponge) having a spongy texture.

Seeding: Sponges were seeded with human or porcine chondrocytes(10⁵-10⁶/20 ul culture medium in a 96 well plate and incubated at 37° C.for 1 hour. Culture medium was added to the well and the spongeincubated 24-48 hours. The sponge was placed into subcutaneous incisionsmade on the back of nude mice.

Implantation procedure: Animals were anesthetized usingketamine-xylazine. Back skin was shaven and cleaned using alcohol. Twoincisions, were made on each side of the back, parallel to the spine. Asubcutaneous pocket or a pocket in the muscle fascia was made from eachincision using blunt dissection. The sponges were implanted in thepockets according to treatment arms. The skin was closed with singlesuture. Each treatment was repeated 5 times and each mouse was implantedwith 4 sponges. See Table 2 hereinbelow.

TABLE 2 Experimental Setup Mouse Left Left Right Right No. proximaldistal proximal distal Tagging 1 1 × 10{circumflex over ( )}5 1 ×10{circumflex over ( )}6 1 × 10{circumflex over ( )}5 1 × 10{circumflexover ( )}6 No tag Human Human Porcine Porcine 2 1 × 10{circumflex over( )}6 1 × 10{circumflex over ( )}5 1 × 10{circumflex over ( )}6 Sponge 1Rt ear Human Human Porcine w/o cells 3 1 × 10{circumflex over ( )}6 1 ×10{circumflex over ( )}5 1 × 10{circumflex over ( )}5 1 × 10{circumflexover ( )}6 1 Lt ear Porcine Porcine Human Human 4 1 × 10{circumflex over( )}5 Sponge 1 × 10{circumflex over ( )}6 1 × 10{circumflex over ( )}5 2Rt ear Porcine w/o cells Human Human 5 1 × 10{circumflex over ( )}6 1 ×10{circumflex over ( )}6 Sponge 1 × 10{circumflex over ( )}5 2 Lt earHuman Porcine w/o cells Human 6 Sponge Sponge 1 × 10{circumflex over( )}6 1 × 10{circumflex over ( )}5 RT + LT w/o cells w/o cells PorcinePorcine

Induced cartilage formation evaluation: One or four weeks postimplantation the mice were sacrificed and the implants with theirsurrounding tissue retrieved and prepared for histology evaluation. Themicroscopically assessment consists of a complete morphologicaldescription of the implant. Additional analysis include H&E stainingsafranin O, alcian blue and anti-collagen type II staining.

FIG. 7A shows the implantation procedure. FIG. 7B shows the growth ofectopic cartilage derived from a cell embedded sponge (10⁵ cells) on theback of a mouse, FIGS. 8A, 8B and 8C show a hematoxylin-eosin stainedsection of human chondrocytes of a neocartilage plug after one week.FIG. 8A shows a histological section with many cells exhibiting strongstaining of cartilage matrix using toluidine blue and fast red.

FIGS. 8B and 8C show histological sections stained with H&E. Note thegood cell dispersion and the presence of cell matrix surrounding thecells. FIGS. 9A and 9B show a hematoxylin-eosin stained section ofporcine chondrocytes at 40× and 200× magnification, after 4 weeks growthin situ.

The results of this experiment confirmed that the matrix comprisingplasma proteins substantially devoid of plasminogen is an effectivematrix for the formation of cartilage. The matrices are non-immunogenic,non-toxic and support chondrocyte growth and differentiation.

Example 13 Method of Matrix Preparation

Sponges were prepared in two different ways and tested for cellviability and cell dispersion. Both methods comprise the steps ofpreparing the plasma protein and thrombin solutions. One method furthercomprises sequential dispensing of the thrombin and fibrinogen solutionsinto a mold. The second method, “premixing”, requires that the twosolutions be mixed prior to casting into a mold. The resulting spongesare different in terms of their porosity and cell absorptioncapabilities. FIG. 10A shows chondrocyte cell viability on the spongesprepared using the two different methods. Cell viability is similar onboth types of sponges. A difference can be seen in porosity and celldispersion.

FIG. 10B shows cells sitting on the upper layers of a sponge preparedusing the premixing method. FIG. 10C shows cell distribution throughoutthe matrix in a sponge prepared according to the method where thesolutions are cast into the mold sequentially. Certain applications maybenefit one type of sponge over another.

Example 14 Sheep Model of Cartilage Repair

This study was designed to evaluate the capacity of the chondrocyteembedded fibrin matrix of the invention to repair cartilage in a largeanimal model. A total of 20 sheep each weighing about 60-80 kg werechosen. Eight of the animals underwent a chondrocyte harvestingprocedure prior to implantation. The harvested chondrocytes wereexpanded and seeded onto recombinant human fibrin matrices.

Animal housing conditions conformed to applicable laws and regulationsrelating to laboratory animals. The experiments were performed inaccordance with the principles of the local laws for Animal Experiments.The animals were examined for evidence of disease or lameness.Acceptability into the study was contingent on being disease free,clinically sound, and no history of prior use. Osteoarthritis wasexcluded by a preoperative X-ray. The animals were conditioned for anappropriate period of time as determined by the institution. A uniquenumber tattoo and ear tag identified each animal. Animals were assignedto the treatment groups by random allocation of identification numbers.The study design is shown below in table 3:

TABLE 3 # Sheep Treatment Type of matrix 1A-7A untreated untreated 1B-7Bmicrofracture microfracture 1C-4C Matrix alone TEA + X-linked HA 5C-8CMatrix alone Plasminogen free + X-linked HA 9C-12C Matrix alonePlasminogen free + non X-linked HA 1D-4D Matrix + cells Plasminogenfree + X-linked HA 5D-8D Matrix + cells Plasminogen free + non X-linkedHA

Animals were observed daily for general health throughout the course ofthe study. In the unlikely event that an animal will become injured,ill, or moribund, care will be conducted in accordance with currentveterinary medical practice. If warranted for humane reasons, euthanasiawill be conducted in a humane manner according to the guidelines setforth by the AVMA (American Veterinary Medical Association) Panel onEuthanasia (JAVMA, March 2000). The attending veterinarian will performa clinical diagnosis and treatment on the animal if it shows signs ofillness.

Bodyweight measurements were taken from all animals once during thequarantine period, prior to surgery (Day 0) and at the end of the study(Day 112).

Group A. Untreated defects: In 7b animals (14 defects) the chondraldefects were created in the condyle and were left untreated.

Group B. Microfracture: In 7 animals (14 defects) microfracture wasperformed in without further treatment. Four microfractures wereperformed with special awls in each defect until punctuate bleeding wasobserved.

Group C. Fibrin matrix alone: Fibrin matrices comprising TEA and crosslinked hyaluronic acid (X-linked HA) were implanted in 4 sheep (1C-4C).A fibrin matrix prepared from the plasminogen free fibrinogen and eitherX-linked (5C-8C) or non-x-linked HA (9C-12C) were implanted in 4 sheep,each. The matrices were implanted after creating the defects asdescribed below.

Group D Cell bearing fibrin matrix: Fibrin matrices prepared fromplasminogen free fibrinogen and either cross linked hyaluronic acid(X-linked HA, 1D-4D)) or non-X-linked HA (5D-8D) were seeded withchondrocytes, and implanted into the knee defects of 4 sheep, each.

Operation: The left knee joint was sterilely draped and opened by ananteromedial approach under general anaesthesia. The medial condyle wasexposed, and small pieces of cartilage were harvested from the lowweight bearing surfaces of the trochlea and intercondylar notch. Thecartilage was cut superficially with a scalpel to avoid bleeding. Thewound closure was performed in layers. An external plaster fixation forstifle joint and ankle was applied for five days and cage activitylimited to reduce joint loading in order to prevent dislodgement of thepatella. The tissue specimen was diced and washed under sterileconditions and the cells isolated by collagenase following a standarddigestion protocol. The cells were plated in 75 ml flasks (Corning) andincubated at 37° C. Changing of media was performed every other day.After 3 weeks about 200,000 (2×10⁵) cells were seeded on the selectedfibrin matrices and cultivated for 4 days in E-well plates. Thecell-bearing matrices were sterilely transferred to the operation room.The medial condyle of the right knee of the same sheep was exposed.Using a 4.5-mm punch (Smith & Nephew), two defects, 1 and 2.5 cm distalfrom the intercondylar notch, were made in the medial condyle of thefemur. The defects were outlined with the dermal punch down to thesubchondral bone and the cartilage was removed with small curettes. Anattempt will be made to remove all of the articular cartilage by gentlyscraping the calcified cartilage surface. No bleeding should be observedfrom the subchondral bone. The fibrin matrices were fixed into placeusing fibrin glue.

After treatment of the defect, bleeding points of the capsule werestopped by cauterization and wound closure performed in layers. Theexternal plaster fixation was applied for another five days and cageactivity limited to reduce joint loading in order to prevent dislodgmentof the graft and reparative tissue. After removal of the plaster, thesheep were given unrestricted activity in runs, and fed with a balancednutrition twice a day. Until the second postoperative day 2 g cefazolinwas administered thrice daily.

All animals of group C and D were sacrificed at 16 weeks afterimplantation as described below and in Mankin, H. (NEJM (1974)291:1335-1340).

Necropsy: Animals were humanely sacrificed at 16 weeks postoperatively.Bodyweights were recorded immediately prior to sacrifice. Deepanesthesia was induced with a mixture of ketamine-xylazine and thesubject exsanguinated according to the guidelines set forth by the AVMAPanel on Euthanasia (JAVMA, March 2000).

Gross evaluation and sample collection as described in table 4 wasperformed. The articulating surfaces opposing the defect sites wereexamined for any abnormal joint surface. Additionally, gross evaluationsof the knee joints were made to determine the cartilage repair based onprevious scoring criteria listed in Table 5. Femora, patellae, synovium,and popliteal lymph nodes shall be harvested and placed intoappropriately labeled containers. Immediately following tissue harvest,gross morphological examination of the cartilage surface was performedand photographic records made of each specimen.

TABLE 4 Gross Evaluation and Sample Collection Gross Sample PhotographSample Evaluation collection and Score Knee joint (incl. X X Xarticulating defect site)

Gross Morphological Observations: Following collection of the kneejoints, the joints are opened, photographed and the surface of thedefect site scored as indicated in Table 5. The synovial membrane wasexamined for inflammation. Joint fluid was collected and analyzed.

TABLE 5 Scoring Criteria for Gross Morphological EvaluationsCharacteristic Grading Score Edge Integration Full 2 (new tissuerelative to native cartilage) Partial 1 None 0 Smoothness of thecartilage surface Smooth 2 Intermediate 1 Rough 0 Cartilage surface,degree of filling Flush 2 Slight depression 1 Depressed/overgrown 0Color of cartilage, opacity or Transparent 2 translucency of theneocartilage Translucent 1 Opaque 0

Histology and Histological Evaluation: The knees were opened understerile conditions and a culture swab obtained. Synovium was documentedmacroscopically and the defects are photographed and the joint grosslyexamined. The distal femur was removed and placed in 10% neutralbuffered formalin for 12 hours. Areas of trochlea containing the defectsand the harvest sites were dissected and placed into 10% formalin for 4days. The specimens were subsequently placed into a decalcificationsolution (100 g Tritriplex (Epignost, Austria) and 33 gTris-hydroxymethylene-amnomethane (Merck Eurolab, Belgium) per liter)for two to four days at room temperature. The decalcified specimens areembedded in paraffin and cut in a microtome to 5 μm thick sections.

Sections are stained with hematoxylin and eosin (H&E), safranin O/FastGreen, alcian blue and azan for evaluation of tissue types.Immunohistochemistry with antibodies for type I and type II collagens isperformed according to a standard ABC protocol using HRP conjugatedantibodies. Normal healthy ovine cartilage and tendon served ascontrols.

Light microscopy is performed on a Vanox Olympus research microscopeimplementing a histomorphometric method to determine the percentage ofselected tissue types (analySiS). Multiple serial transversehistological sections from the middle portion of the defect areevaluated. The filling of the defect is determined as an area percentageof reparative tissue in the defect, based on the cross-sectional area ina sagittal plane through the center of the lesion. The area of thedefect, of the filling, height and base of the defect, and tissue typeare evaluated. The tissue types are characterized as follows: 1. fibroustissue 2. transitional tissue 3. hyaline tissue and 4. articularcartilage. Semiquantitative analysis of the defect and adjacent tissueare done according standard scores adapted from O'Driscoll, Pineda andFrenkel.

Example 15 Human Clinical Trial

A feasibility study to evaluate the safety and performance of the fibrinmatrices of the invention in the treatment of chronic cartilage defectsof the femoral condyle has been submitted and approved (Ethics Committeeof the Vienna University Hospital).

A phase I, non-randomized, open label, safety study using a fibrinmatrix or a cell-bearing fibrin matrix prepared using plasminogen-freefibrinogen and autologous chondrocyte in patients is performed. Patientsmeeting the entrance criteria will undergo an arthroscopic procedure toconfirm diagnosis and to harvest a biopsy for the growth of chondrocytesfor future transplantation. Three to six weeks following cell harvest,patients will be hospitalized for surgery. After surgery, patients willbe monitored for safety as follows: during 5-7 days hospitalization;after discharge at week 2 and week 6, and performance evaluation at week12, month 6, and month 12.

The primary end is to evaluate the safety of a cell-bearing matrix ofthe invention, wherein the matrix serves as a scaffold for the seedingand transplantation of autologous chondrocytes in the treatment of achronic cartilage condyle lesion.

The secondary endpoint is to evaluate the performance of a cell-bearingmatrix in restoring function, as measured by an improvement in: MRIscores, Quality of life questionnaire, Joint function score. The safetyparameters will include vital signs, serum chemistry, hematology andsystemic and local adverse events. All parameters including patientinclusion and exclusion criteria and patient withdrawal criteria arepresented.

Example 16 One-Step Procedure for Treating Damaged Cartilage: Suitablefor Arthroscopy or Hemi-Arthrotomy

Autologous chondrocyte implantation has proven clinically effective inrestoring hyaline-like cartilage to isolated chondral defects of theknee. The present therapies include three major steps:

1. Diagnostic Arthroscopy and biopsy of healthy cartilage.

2. Cultivation of cells.

3. Injection of cultured chondrocytes into the lesion under a periostealflap, which is taken from the tibia and sutured over the lesion.

The disadvantages of the technique include the need for two separatesurgical procedures, the requirement for a second site surgery toisolate a periosteal flap and the tendency for cartilage overgrowth dueto the presence of the flap. An improved variation of this techniqueprovides implant of the matrix of the present invention. A lesstraumatic method is presented herein, wherein the patient undergoes asingle surgical procedure for cartilage repair.

Procedure: A patient with a cartilage defect may donate autologousplasma several days prior to the arthroscopy or hemi-arthrotomy. Blood(approximately 100-250 ml) is drawn and plasma proteins are purified,removing plasminogen. A plasma protein matrix, or several matrices, isprepared, labeled and stored aseptically until the day of the surgery.

Optionally, on the day of the surgery, cartilage from the patient'sjoint is removed, cut into small pieces and placed in a test tubecontaining collagenase, hyaluronidase or other cartilage degradingenzymes, or combinations thereof.

The surgeon treats the defective region of the joint by removing damagedtissue, cleansing and preparing the area for an implant. The preparedmatrix is removed from its container and cut to fit the defectivedomain. Following approximately 20-30 minutes of enzymatic treatment,the cells and small pieces of cartilage are spun down in a tabletopcentrifuge, rinsed in PBS and resuspended in a small amount (50 ul-1000ul) of PBS. The surgeon seeds the cells onto the sponge, in situ.Alternatively, the cells are absorbed into the sponge and thecell-bearing sponge implanted into the defective joint region.Optionally, extracellular matrix degrading enzymes and or otherbioactive agents including growth factors and/or anti-inflammatorycompounds are added to the sponge. In certain instances the surgeon willplace a dry sponge directly onto the injured area, optionally add enzymesolution to said sponge and place a second, cell-bearing sponge on topof the first sponge. The joint is closed and is treated as customary foran arthroscopic or hemi-arthrotomy procedure and the patient is releasedto recover.

Kit

A kit comprising the components useful for practicing the method of theinvention, will allow for the convenient practice of the method of theinvention in a surgical setting. In one embodiment, a kit of theinvention will provide sterile components suitable for easy use in thesurgical environment including, sterile solutions (saline, enzymes) asterile, cell-free matrix material suitable for supporting autologouschondrocytes that are to be implanted into an articular joint surfacedefect and instructions for use.

Example 17 Bone Repair Model

The plasma protein matrix of the present invention is useful for thetreatment of bone defects including osteotomy, particularly innon-weight bearing regions of the skeleton.

Suitable animal models are used to create bilateral osteotomies todemonstrate the efficacy of the present invention. In an exemplaryrabbit model a 4-6 mm osteotomy is created in New Zealand Rabbits incompliance with the Animal Care Committee. The ulna is chosen because itis only slightly weight-bearing and allows the creation of a bone defectwithout requiring a cast or other immobilization treatment. In addition,this gap constitutes a spontaneously healing defect that allows theevaluation of the tested agent. The primary indices of fracture healingare accelerated duration of healing and callus formation. The testcompounds consist of matrices of the invention and control matrices.

Surgery procedure: Animals are anesthetized according to standardprotocol. Gap formation is performed in the mid ulna bone. A sponge ofthe invention is placed into the gap area in each limb and the fractureis closed. Animals are treated with analgesic for 3 days post operation.The duration of the experiment is 6 weeks.

Healing time and quality assessment: X-ray grading provides fracturehealing status assessment. Rabbits are X-rayed every other week for 5weeks after surgery. X-rays are scored by two orthopedic surgeons in ablinded manner according to a standard grading scale protocol.

Quality evaluation: at the end of the experiment, rabbits are sacrificedand fracture area is sent for histological and mechanical strengthevaluation. Histology is scored by a pathologist for evaluation ofhistological changes during the healing process using standard stainingmethods, using hematoxylin and eosin (H&E) for cytoplasm and nucleus.Indigo-carmin staining is also applied for detection of newly generatedcallus. Mechanical strength evaluation is performed using the “4 pointsbending” method.

The treatments groups are: sham osteotomy, osteotomy treated with fibrinsponge alone, osteotomy treated with fibrin sponge comprisingglycosaminoglycan, osteotomy treated with a fibrin sponge comprisingglycosaminoglycan, optional heparin growth factors.

Another example of an animal model for bone repair is presented in Cooket al., (Am J. Vet Res 64:2-20, 2003).

While the present invention has been particularly described, personsskilled in the art will appreciate that many variations andmodifications can be made. Therefore, the invention is not to beconstrued as restricted to the particularly described embodiments,rather the scope, spirit and concept of the invention will be morereadily understood by reference to the claims which follow.

1. A method of treating diseased or injured tissue, which comprisesimplanting into the tissue at a site of the disease or injury a porousfreeze-dried fibrin matrix formed from plasma proteins comprisingfibrinogen cleaved by the action of thrombin at varying concentrationssufficient to cleave said fibrinogen and Factor XIII, the matrix havingless than 10% residual moisture and being devoid of exogenousanti-fibrinolytic agents, plasminogen and of organic chelating agents.2. The method according to claim 1, wherein said plasma proteinscomprise partially purified plasma proteins.
 3. The method according toclaim 1, wherein said plasma proteins comprise less than 20% ofplasminogen normally present in plasma.
 4. The method according to claim1, wherein said porous freeze-dried fibrin matrix further comprises atleast one additive, wherein the at least one additive is aglycosaminoglycan and at least one bioactive agent, wherein the at leastone bioactive agent is a growth factor.
 5. The method according to claim4, wherein the glycosaminoglycan is crosslinked or non-crosslinkedhyaluronic acid.
 6. The method according to claim 4, wherein the growthfactor is a fibroblast growth factor.
 7. The method according to claim6, wherein the fibroblast growth factor is selected from the groupconsisting of FGF2, FGF4, FGF9, and FGF18.
 8. The method according toclaim 1, wherein said porous freeze-dried fibrin matrix furthercomprises cells.
 9. The method according to claim 8, wherein the cellsare selected from the group consisting of: stem cells, progenitor cells,chondrocytes, osteoblasts, hepatocytes, and mesenchymal, endothelial,epithelial, urothelial, endocrine, neuronal, pancreatic, renal andocular cell types.
 10. The method according to claim 1, wherein at leastone of said plasma proteins is autologous or at least one of said plasmaproteins is recombinant.
 11. The method according to claim 1, whereinthe matrix is prepared by a process comprising the steps of: providing athrombin solution and a plasma protein solution wherein the plasmaprotein solution comprises fibrinogen and Factor XIII and is devoid ofexogenous anti-fibrinolytic agents and of organic chelating agents;introducing the thrombin solution and the plasma protein solution to asolid receptacle or mold in the presence of calcium ions; incubatingunder conditions appropriate to achieve clotting; freezing the clottedmixture; and lyophilizing the clotted mixture, to obtain a porousfreeze-dried fibrin matrix.
 12. A method of treating diseased or injuredtissue, which comprises implanting to the tissue at a site of thedisease or injury a porous freeze-dried fibrin matrix formed from plasmaproteins comprising fibrinogen cleaved by the action of thrombin atvarying concentrations sufficient to cleave said fibrinogen and FactorXIII, the matrix having less than 10% residual moisture and being devoidof exogenous anti-fibrinolytic agents and of organic chelating agents,wherein said plasma proteins comprise partially purified plasma proteinsthat are devoid of plasminogen.
 13. The method according to claim 12,wherein said plasma proteins comprise less than about 5% of plasminogennormally present in plasma.
 14. The method according to claim 12,wherein said porous freeze-dried fibrin matrix further comprises atleast one additive, wherein the at least one additive is aglycosaminoglycan and at least one bioactive agent, wherein the at leastone bioactive agent is a growth factor.
 15. The method according toclaim 14, wherein the glycosaminoglycan is crosslinked ornon-crosslinked hyaluronic acid.
 16. The method according to claim 14,wherein the growth factor is a fibroblast growth factor.
 17. The methodaccording to claim 16, wherein the fibroblast growth factor is selectedfrom the group consisting of FGF2, FGF4, FGF9, and FGF18.
 18. The methodaccording to claim 12, wherein said porous freeze-dried fibrin matrixfurther comprises cells.
 19. The method according to claim 18, whereinthe cells are selected from the group consisting of: stem cells,progenitor cells, chondrocytes, osteoblasts, hepatocytes, andmesenchymal, endothelial, epithelial, urothelial, endocrine, neuronal,pancreatic, renal and ocular cell types.
 20. The method according toclaim 12, wherein at least one of said plasma proteins is autologous orat least one of said plasma proteins is recombinant.